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Alterations in ribosome biogenesis cause specific defects in C. elegans hermaphrodite gonadogenesis
Abstract
Ribosome biogenesis is a cell-essential process that influences cell growth, proliferation, and differentiation. How ribosome biogenesis impacts development, however, is poorly understood. Here, we establish a link between ribosome biogenesis and gonadogenesis in Caenorhabditis elegans that affects germline proliferation and patterning. Previously, we determined that pro-1(+)activity is required in the soma--specifically, the sheath/spermatheca sublineage--to promote normal proliferation and prevent germline tumor formation. Here, we report that PRO-1, like its yeast ortholog IPI3, influences rRNA processing. pro-1 tumors are suppressed by mutations in ncl-1 or lin-35/Rb, both of which elevate pre-rRNA levels. Thus, in this context, lin-35/Rb acts as a soma-autonomous germline tumor promoter. We further report the characterization of two additional genes identified for their germline tumor phenotype, pro-2 and pro-3, and find that they, too, encode orthologs of proteins involved in ribosome biogenesis in yeast (NOC2 and SDA1, respectively). Finally, we demonstrate that depletion of additional C. elegans orthologs of yeast ribosome biogenesis factors display phenotypes similar to depletion of progenes. We conclude that the C. elegans distal sheath is particularly sensitive to alterations in ribosome biogenesis and that ribosome biogenesis defects in one tissue can non-autonomously influence proliferation in an adjacent tissue.
Supplementary resources (2)
... Many aspects of nucleolar function and ribosome biogenesis are conserved within eukaryotic organisms, from yeast to humans. The model organism C. elegans contains many genes that are involved in ribosome biogenesis, such as rsks-1 encoding a homolog of S6K that positively regulates several steps in ribosome biogenesis 13 , as well as the pro-1, pro-2, and pro-3 genes, which have been implicated in rRNA processing and ribosome assembly 14 . In the present study, we first identify a mutation in rrp-8, which encodes an ortholog of rRNA processing 8 (RRP8) and leads to an impaired ribosome profile and elevated lipid accumulation. ...
... To further confirm the above results, we performed northern blotting to analyze rRNA processing. The processed rRNA intermediates were analyzed based on previous reports 14,22 . To detect the levels of pre-RNA (a) and rRNA intermediates (b, b1, c, and c1) in WT and rrp-8(kun54) mutant worms, we designed three probes (1)(2)(3) corresponding to distinct regions of the pre-RNA (Fig. 2e) following the report by Voutev et al. 23 . ...
... In C. elegans, the pro-1, pro-2, and pro-3 genes have been implicated in rRNA processing and ribosome assembly 14 . Similarly, two available mutants, pro-2(na27) and pro-3(ar226), also showed increased fat contents (58.45 ± 0.51 and 57.36 ± 0.39, respectively) compared with WT worms according to the TLC/ GC analysis (Fig. 3a). ...
... Many aspects of nucleolar function and ribosome biogenesis are conserved within eukaryotic organisms, from yeast to humans. The model organism C. elegans contains many genes that are involved in ribosome biogenesis, such as rsks-1 encoding a homolog of S6K that positively regulates several steps in ribosome biogenesis 13 , as well as the pro-1, pro-2, and pro-3 genes, which have been implicated in rRNA processing and ribosome assembly 14 . In the present study, we first identify a mutation in rrp-8, which encodes an ortholog of rRNA processing 8 (RRP8) and leads to an impaired ribosome profile and elevated lipid accumulation. ...
... To further confirm the above results, we performed northern blotting to analyze rRNA processing. The processed rRNA intermediates were analyzed based on previous reports 14,22 . To detect the levels of pre-RNA (a) and rRNA intermediates (b, b1, c, and c1) in WT and rrp-8(kun54) mutant worms, we designed three probes (1)(2)(3) corresponding to distinct regions of the pre-RNA (Fig. 2e) following the report by Voutev et al. 23 . ...
... In C. elegans, the pro-1, pro-2, and pro-3 genes have been implicated in rRNA processing and ribosome assembly 14 . Similarly, two available mutants, pro-2(na27) and pro-3(ar226), also showed increased fat contents (58.45 ± 0.51 and 57.36 ± 0.39, respectively) compared with WT worms according to the TLC/ GC analysis (Fig. 3a). ...
The primary function of the nucleolus is ribosome biogenesis, which is an extremely energetically expensive process. Failures in ribosome biogenesis cause nucleolar stress with an altered energy status. However, little is known about the underlying mechanism linking nucleolar stress to energy metabolism. Here we show that nucleolar stress is triggered by inactivation of RSKS-1 (ribosomal protein S6 kinase), RRP-8 (ribosomal RNA processing 8), and PRO-2/3 (proximal proliferation), all of which are involved in ribosomal RNA processing or inhibition of rDNA transcription by actinomycin D (AD), leading to excessive lipid accumulation in Caenorhabditis elegans. The transcription factor PHA-4/FoxA acts as a sensor of nucleolar stress to bind to and transactivate the expression of the lipogenic genes pod-2 (acetyl-CoA carboxylase), fasn-1 (fatty acid synthase), and dgat-2 (diacylglycerol O-acyltransferase 2), consequently promoting lipid accumulation. Importantly, inactivation of pha-4 or dgat-2 is sufficient to abolish nucleolar stress-induced lipid accumulation and prolonged starvation survival. The results revealed a distinct PHA-4-mediated lipogenesis pathway that senses nucleolar stress and shifts excessive energy for storage as fat.
... We UV-cross-linked proteins to mRNAs in vivo and performed immunopurification (IP) of endogenously GFP-tagged proteins from cell lysates under stringent conditions. We then assessed the abundance of proteins and bound mRNAs via GFP and hybridization with fluorescently labeled oligo(dT) 25 , respectively (Fig. 4). As expected, we detected poly(A) mRNAs in the IP of three known mRBPs (Gis2, Khd1 and Pab1) but observed no RNA-dependent associations with untagged wild-type control cells or Ras1p, a GTPase not known to bind RNA. ...
... The presence of mRNAs in poly(A) eluates was controlled by reverse-transcription (RT)-PCR (data not shown). To capture polyadenylated RNAs for mRBPome analysis, 1 mg of oligo(dT) 25 Dynabeads (Life Technologies, 61011) was equilibrated in lysis buffer, mixed with the extracts (~5 mg) and incubated on a shaker for 10 min at room temperature (RT). The beads were collected with a magnet, and the supernatant was recovered for repeat incubations (described below). ...
... The poly(A) RNA was eluted from beads in 60 µl of 10 mM Tris-HCl, pH 7.5, at 80 °C for 2 min and collected. The entire procedure was repeated twice by reapplying the supernatant to the oligo(dT) 25 beads, which were recovered after elution and washed three times with lysis buffer before reapplication. The three sequential eluates were combined and concentrated to 70 µl in a 0.5 ml Microcon-10kDa centrifugal filter unit with an Ultracel-10 membrane (Millipore, MRCPRT010). ...
RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae. Strikingly, most proteins composing mRBPomes, including components of early metabolic pathways and the proteasome, are evolutionarily conserved between yeast and C. elegans. We speculate, on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs, that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'.
... colleagues [52]. Interestingly, the conditions that provoked this Pro phenotype were mutations or RNAi knockdowns of genes involved in rRNA processing [53]. Formation of the proximal tumours in these conditions involves events in early germ line development (as many germ cell proliferation phenotypes do [e.g., 54,55]) and is likely a result of spatiotemporal mismatch between early germ cells and the signalling environment [56]. ...
... Defects of certain early steps of eukaryotic rRNA processing can lead to accumulation of pre-rRNA or to processing by alternative routes, eventually resulting in a characteristic pattern of processing intermediates that can be separated by electrophoresis. For instance, pro-1 mutants in C. elegans differently process rRNA at the internal transcribed spacer its2, similar to yeast mutants of its homolog Ipi3 [53]. ...
... There is a high number of factors involved specifically in the synthesis of either of the two subunits as well as factors needed for both [61]. Interestingly, several worm mutants of rRNA processing factors had been isolated from one genetic screen, based on their conditional proximal proliferation (Pro) phenotype described above [52,53]. We thus looked for apoptotic defects in the viable Pro mutants pro-2(na27) and pro-3(ar226). ...
Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.
... phb-1, and phb-2) lead to arrest as sterile adults. In a number of cases, the identified suppressor genes have previously been shown to be required for late larval development or fertility, thereby precluding suppression to viable or fertile adults (Table S1) (Hanazawa et al. 2004;Checchi and Kelly 2006;Voutev et al. 2006;Rodenas et al. 2009). ...
... Human metaxin-2/unknown function/novel For this class, which includes pro-2, suppressed lin-35; slr-2 mutants arrested as sterile adults ( Figure S1). The roles of these genes in ribosome biogenesis are inferred both from sequence homology to well-characterized orthologs in yeast and from studies in C. elegans showing that mutations in a subset of these genes lead to defects in rRNA processing (Bousquet-Antonelli et al. 2000;Vanrobays et al. 2001;Galani et al. 2004;Voutev et al. 2006). In yeast, Gar1/Y66H1A.4 ...
... Y56A3A.18, and Y48A6C.4) form proximal germline tumors resulting from defects in the developing somatic gonad (Killian and Hubbard 2004;Voutev et al. 2006). The strongest suppressor identified by our screen, C16A3.4, ...
The Caenorhabditis elegans pRb ortholog, LIN-35, functions in a wide range of cellular and developmental processes. This includes a role of LIN-35 in nutrient utilization by the intestine, which it carries out redundantly with SLR-2, a zinc-finger protein. This and other redundant functions of LIN-35 were identified in genetic screens for mutations that display synthetic phenotypes in conjunction with loss of lin-35. To explore the intestinal role of LIN-35, we conducted a genome-wide RNA-interference-feeding screen for suppressors of lin-35; slr-2 early larval arrest. Of the 26 suppressors identified, 17 fall into three functional classes: (1) ribosome biogenesis genes, (2) mitochondrial prohibitins, and (3) chromatin regulators. Further characterization indicates that different categories of suppressors act through distinct molecular mechanisms. We also tested lin-35; slr-2 suppressors, as well as suppressors of the synthetic multivulval phenotype, to determine the spectrum of lin-35-synthetic phenotypes that could be suppressed following inhibition of these genes. We identified 19 genes, most of which are evolutionarily conserved, that can suppress multiple unrelated lin-35-synthetic phenotypes. Our study reveals a network of genes broadly antagonistic to LIN-35 as well as genes specific to the role of LIN-35 in intestinal and vulval development. Suppressors of multiple lin-35 phenotypes may be candidate targets for anticancer therapies. Moreover, screening for suppressors of phenotypically distinct synthetic interactions, which share a common altered gene, may prove to be a novel and effective approach for identifying genes whose activities are most directly relevant to the core functions of the shared gene.
... Mutations in several genes were identified that cause both a reduction in larval germline proliferation and a proximal tumor. Through a series of events, inadequate larval germline proliferation can set up inappropriate somagermline contacts that, counter-intuitively, cause the formation of a proximal germline tumor ( Hubbard, 2004, 2005;Voutev et al., 2006). Briefly, inadequate larval proliferation delays differentiation since the gonad does not elongate properly and germ cells remain in proximity to the DTC. ...
... For example, PRO-1, PRO-2, and PRO-3 are related to yeast IPI3, NOC2, and SDA1, proteins involved in rRNA processing, a nucleolus-nucleus complex, and later stages of ribosome biogenesis, respectively. Molecular analysis confirmed a role for pro-1 in worm rRNA processing ( Killian and Hubbard, 2004;Voutev et al., 2006). Genetic mosaic studies narrowed the focus of activity of the pro-1 gene to the SS lineage ( Killian and Hubbard, 2004), and sheath cell defects were observed after reducing the activity of several ribosome biogenesis genes ( Voutev et al., 2006). ...
... Molecular analysis confirmed a role for pro-1 in worm rRNA processing ( Killian and Hubbard, 2004;Voutev et al., 2006). Genetic mosaic studies narrowed the focus of activity of the pro-1 gene to the SS lineage ( Killian and Hubbard, 2004), and sheath cell defects were observed after reducing the activity of several ribosome biogenesis genes ( Voutev et al., 2006). Taken together with the cell ablation studies that pinpoint Sh1 as the source of a critical germline proliferation-promoting cell-cell interaction, these data suggest that optimal ribosome biogenesis in the distal pair sheath cells, Sh1, is important for proper larval germline proliferation. ...
Caenorhabditis elegans boasts a short lifecycle and high fecundity, two features that make it an attractive and powerful genetic model organism. Several recent studies indicate that germline proliferation, a prerequisite to optimal fecundity, is tightly controlled over the course of development. Cell proliferation control includes regulation of competence to proliferate, a poorly understood aspect of cell fate specification, as well as cell-cycle control. Furthermore, dynamic regulation of cell proliferation occurs in response to multiple external signals. The C. elegans germ line is proving a valuable model for linking genetic, developmental, systemic, and environmental control of cell proliferation. Here, we consider recent studies that contribute to our understanding of germ cell proliferation in C. elegans. We focus primarily on somatic control of germline proliferation, how it differs at different life stages, and how it can be altered in the context of the life cycle and changes in environmental status.
... In addition, LIN-35 redundantly controls organ morphogenesis (Fay et al., 2003; Bender et al., 2007; Mani and Fay, 2009), cell fate specification (Bender et al., 2004a), maintenance of organ homeostasis (Kirienko et al., 2008 ), and other developmental processes essential for fertility and viability (Cui et al., 2004; Cardoso et al., 2005; Chesney et al., 2006; Ceron et al., 2007). LIN-35 also plays a role in controlling ribosome biogenesis (Voutev et al., 2006), promoting the expression of transgenes (Hsieh et al., 1999 ), and inhibiting the RNAi response in somatic tissues (Wang et al., 2005; Lehner et al., 2006). Notably, many of these reported functions have correlates with some of the proposed tumor-suppressing functions of mammalian pocket proteins. ...
... More recently, several genes affecting ribosome biogenesis have been identified in screens for mutants that develop proximal tumors. Among these are pro1–3, which encode orthologs of yeast proteins that regulate ribosome activity at the level of rRNA processing and nucleolar transport (Killian and Hubbard, 2004; Voutev et al., 2006). Furthermore, PRO-1 is specifically required within the sheath cell lineage to inhibit the formation of germline tumors (Killian and Hubbard, 2004). ...
... These results indicate that gross disruption of normal metabolic processes within sheath cells can lead to nonautonomous defects in the proximal germline. This conclusion is further supported by the finding that ablation of Sh2–Sh5 can fully revert germline tumorigenesis in pro-1 mutants (Killian and Hubbard, 2005) and that partial suppression of pro-1 tumors can also be achieved through secondary mutations that increase prerRNA levels (Voutev et al., 2006 ). Of interest , one of the mutations found to suppress pro-1 tumors is lin-35/Rb, which functions as a negative regulator of POL I rRNA transcription (Hannan et al., 2000; Pelletier et al., 2000; Voutev et al., 2006 ). ...
Although now dogma, the idea that nonvertebrate organisms such as yeast, worms, and flies could inform, and in some cases even revolutionize, our understanding of oncogenesis in humans was not immediately obvious. Aided by the conservative nature of evolution and the persistence of a cohort of devoted researchers, the role of model organisms as a key tool in solving the cancer problem has, however, become widely accepted. In this review, we focus on the nematode Caenorhabditis elegans and its diverse and sometimes surprising contributions to our understanding of the tumorigenic process. Specifically, we discuss findings in the worm that address a well-defined set of processes known to be deregulated in cancer cells including cell cycle progression, growth factor signaling, terminal differentiation, apoptosis, the maintenance of genome stability, and developmental mechanisms relevant to invasion and metastasis.
... Because some yeast components involved in rRNA syntheis play dual roles in rRNA biogenesis and cell cycle control, we we wanted to determine if the nol-10(rr45) growth phenotype caused ribosomal defects independent of its role in cell cycle (Bernstein et al., 2007). Mutants that cause the resumption of cell proliferation in the proximal germ line, namely pro-1, reduces the level of mature 26s rRNA, and the hyperproliferation phenotype can be suppressed by the increased precursor rRNA levels typical of lin-35 or ncl-1 mutants (Voutev et al., 2006). However, as previously reported, the double mutant lin-35(rr33); nol-10(rr45) is synthetic lethal, suggesting that they might act in the same pathway or in parallel pathways that impinge on the same cellular process. ...
... To determine if the intestinal cell cycle defect observed in nol-10(rr45) is common to genes required in ribosomal biogenesis, we used RNAi to reduce the gene activity of pro-1, pro-3 and rbd-1, three genes shown to be required for appropriate RNA biogenesis (Voutev et al., 2006). Although feeding RNAi of any of these three genes strongly induces a growth defect phenotype typical of ribosomal genes, the effects on the intestinal division were variable (Table 4.1). ...
... The nol-10(rr45) also shows a post-embryonic intestinal cell cycle defect so it prompted us to determine if the defects observed in rRNA transcription are the cause of the intestinal cell cycle defects. In order to address this question, we looked at the intestinal phenotype of two genes previously shown to be required in rRNA biogenesis, pro-1 and pro-3 (Voutev et al., 2006). RNAi against these two genes causes a strong growth phenotype as expected from genes required in rRNA synthesis but we also detect a small, but significant increases in the number of intestinal nuclei. ...
The development of the nematode C. elegans is invariant allowing investigators to use genetic analysis in this model system to identify components of many critical developmental pathways. The Roy laboratory uses this strategy to identify genes that control cell division throughout the course of development. My work focusses on the intestinal lineage due to their typical cell division pattern. Using genetic analysis, I have characterized the role of genes that affect the transition from mitosis to karyokinesis and finally endoreplication that occurs in the intestinal cells post-embryonically during the first larval stage. Five mutants that were isolated are grouped into classes that have more (rr33 and rr45) or less (rr42, rr43 and rr44) intestinal nuclei than wild-type. I cloned both mutants with more intestinal nuclei (rr33 and rr45) to show that they encode lin-35, the homologue of the Retinoblastoma gene and a highly conserved gene referred to as nol-10 respectively. I showed that lin-35 interacts with RNAi pathway components to properly repress genes required for this transition. I also found that met-2, the homologue of the Suv39h methyl transferase as well as the heterochromatin protein-like hpl-1 and hpl-2 are required to maintain the repression of these loci. Moreover, during my characterization of the typical cell cycle arrest observed during the dauer stage, I found that the Notch ligand lag-2 is specifically expressed in 6 Inter Labial neurons 2 (IL2) at the onset and throughout the dauer stage. I demonstrated that this expression pattern reflects the requirement of the canonical Notch pathway to properly maintain the dauer stage and specific expression of the Notch receptor glp-1 in a subset of neurons is able to rescue the defect in dauer maintenance in glp-1 mutants. Therefore, the Notch pathway is not required for neuronal cell fate specification since there is no cell division at this stage but rather serves a novel function that may involve neuronal remodeling or effects on some physiological process. Puisque son développement ne varie pas, le nématode C. elegans est un organisme pouvant servir de modèle génétique pour identifier des gènes requis dans bon nombre de processus. De ce fait, le laboratoire du Dr Roy utilise cet organisme pour identifier des gènes qui régulent la division cellulaire durant son développement. L'isolement de 5 mutants, qui influencent la division cellulaire de l'intestin, m'a permis de caractériser le rôle de ces gènes dans la transition de la mitose vers la karyokinèse, puis vers l'endoréplication. Cette transition est spécifique aux cellules intestinales à la fin du premier stade larvaire du développement post-embryonnaire. Ces cinq mutants ont été groupés soit dans la classe de mutants avec plus de noyaux intestinaux que la souche sauvage (rr33 et rr45), soit dans celle en contenant moins (rr42, rr43 et rr44). Le clonage des mutants contenant plus de noyaux intestinaux m'a permis d'observer l'influence de la mutation rr33 sur le gène lin-35, l'homologue du gène du rétinoblastome chez l'humain, et de rr45 sur le gène nol-10. Je démontre que lin-35 interagit avec les composantes de la voie RNAi afin de permettre la répression des gènes requis pour la transition du cycle cellulaire des cellules intestinales. Mes résultats permettent également d'observer le rôle de met-2, un homologue de la méthyltransférase Suv39h et de deux protéines hétérochromatiques hpl-1 et hpl-2, dans le maintien de cette répression. De plus, j'ai pu constater que, durant ma caractérisation de l'arrêt du cycle cellulaire propre au stade dauer, le ligand Notch lag-2 est spécifiquement exprimé au début et durant tout ce stade dans 6 neurones Inter Labial 2 (IL2). J'ai pu démontrer que l'expression de ce ligand correspond au besoin de la voie de signalement canonique Notch pour maintenir ce stade de développement et aussi que l'expression du récepteur Notch glp-1 dans les neurones empêche le défaut de maintien dû à une mutation dans glp-1 de survenir. Finalement, la voie de signalement Notch n'est pas requise pour la spécification de la destinée des cellules puisqu'il n'y a pas de division cellulaire durant ce stade, mais remplit plutôt une nouvelle fonction, soit dans le remaniement neuronal, soit dans un processus physiologique.
... Among them is GLD-1, a maxi-KH/STAR RNA-binding protein functioning as a translational repressor [32][33][34][35]. Another example is PRO-1, a conserved WDrepeat protein implicated in rRNA processing [36]. Without these factors, germ cells develop into highly penetrant tumors, although found in different gonadal regions (Fig 4A). ...
... Thus, reducing translational output, by removing METL-13, could explain the partial suppression of the tumorous proliferation observed upon the depletion of GLD-1. Whether the same applies to pro-1 tumors is less clear, as their etiology is less well understood [36]. ...
The methyltransferase-like protein 13 (METTL13) methylates the eukaryotic elongation factor 1 alpha (eEF1A) on two locations: the N-terminal amino group and lysine 55. The absence of this methylation leads to reduced protein synthesis and cell proliferation in human cancer cells. Previous studies showed that METTL13 is dispensable in non-transformed cells, making it potentially interesting for cancer therapy. However, METTL13 has not been examined yet in whole animals. Here, we used the nematode Caenorhabditis elegans as a simple model to assess the functions of METTL13. Using methyltransferase assays and mass spectrometry, we show that the C. elegans METTL13 (METL-13) methylates eEF1A (EEF-1A) in the same way as the human protein. Crucially, the cancer-promoting role of METL-13 is also conserved and depends on the methylation of EEF-1A, like in human cells. At the same time, METL-13 appears dispensable for animal growth, development, and stress responses. This makes C. elegans a convenient whole-animal model for studying METL13-dependent carcinogenesis without the complications of interfering with essential wild-type functions.
... In fact, reduced overall protein synthesis was shown to extend lifespan in several model organisms, including the nematode Caenorhabditis elegans (Hansen et al., 2007;Pan et al., 2007;Syntichaki et al., 2007). Although some evidence indicates that a link between ribosome biogenesis and gonadogenesis in C. elegans may exist (Voutev et al., 2006), the precise relationship between these pathways in multicellular organisms is still poorly understood. It is conceivable that the optimal function of ribosomes, which requires the presence of ribosomal RNA (rRNA) and ribosomal protein (r-protein) modifications, is monitored by the cell at several stages during development. ...
... It is conceivable that the optimal function of ribosomes, which requires the presence of ribosomal RNA (rRNA) and ribosomal protein (r-protein) modifications, is monitored by the cell at several stages during development. Thus, introduction of these modifications might participate in the control of cell fate and cell-cell interactions during development (Hokii et al., 2010;Voutev et al., 2006). ...
Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m ⁵ C at position C2381 on the 26S rRNA in Caenorhabditis elegans . Here, we show that NSUN-1 is writing the second known 26S rRNA m ⁵ C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.
... Importantly, the distance reached by the DTC is the combined result of the intrinsic DTC migration plus the force of additional biomass from proliferating germ cells within the gonad (imagine air filling a long balloon). Anatomical and genetic manipulations Hubbard 2004, 2005;Voutev et al. 2006;Voutev and Hubbard 2008;McGovern et al. 2009) together with computer modeling studies (Atwell et al. 2015;Hall et al. 2015) underscore the importance of these combined spatial and temporal aspects of gonad development for germline patterning (see Appendix). Since alterations in larval somatic or germline morphogenesis can lead to surprising outcomes in the adult, interpretation of adult phenotypes requires consideration of events in all preceding stages. ...
... PRO-1 is a WD repeat containing protein that, like its yeast ortholog IPI3, is important for removal of the internal transcribed spacer two from preribosomal RNA. Other aspects of ribosome biogenesis are likely also important since loss of PRO-2 NOC2L and PRO-3 SDA1 affect the sheath lineage and/or cause a Pro phenotype (Killian and Hubbard 2004;Voutev et al. 2006). ...
Stem cell systems regulate tissue development and maintenance. The germline stem cell system is essential for animal reproduction, controlling both the timing and number of progeny through its influence on gamete production. In this review, we first draw general comparisons to stem cell systems in other organisms, and then present our current understanding of the germline stem cell system in Caenorhabditis elegans In contrast to stereotypic somatic development and cell number stasis of adult somatic cells in C. elegans, the germline stem cell system has a variable division pattern, and the system differs between larval development, early adult peak reproduction and age-related decline. We discuss the cell and developmental biology of the stem cell system and the Notch regulated genetic network that controls the key decision between the stem cell fate and meiotic development, as it occurs under optimal laboratory conditions in adult and larval stages. We then discuss alterations of the stem cell system in response to environmental perturbations and aging. A recurring distinction is between processes that control stem cell fate and those that control cell cycle regulation. C. elegans is a powerful model for understanding germline stem cells and stem cell biology.
... As no notable defects were found in the spermatheca in dao-5(ok542) hermaphrodites and males (data not shown), the fertility defect of dao-5 mutants seems only attributed to the oogenesis. The gonadal defects observed in the absence of DAO-5 are also reminiscent of the phenotypes observed in pro (proximal tumor in gonad) mutants, whose ribosome production is also disrupted, 40 although their underlying mechanism may differ. Sheath cells, a specialized somatic tissue consisting of five pairs of flat cells that surround the germline, are crucial to coordinate germline development. ...
... 41 Impairments of gonadogenesis that occurred in pro-1, pro-2, and pro-3 mutants are considered the result of the affected sheath cells in terms of lower cell number, particularly, in a nonautonomous manner. 40 We, however, are inclined to the view that the gonadal defects observed in DAO-5 deficiency are cell autonomous based on two lines of observation. First, the number of sheath cells around the gonad was not reduced in the absence of DAO-5 (data not shown). ...
Human diseases of impaired ribosome biogenesis resulting from disruption of rRNA biosynthesis or loss of ribosomal components are collectively described as 'ribosomopathies'. Treacher Collins syndrome (TCS), a representative human ribosomopathy with craniofacial abnormalities, is attributed to mutations in the tcof1 gene that has a homologous gene called nopp140. Previous studies demonstrated that the dao-5 (dauer and aged animal overexpression gene 5) of Caenorhabditis elegans is a member of nopp140 gene family and plays a role in nucleogenesis in the early embryo. Here, we established a C. elegans model for studying Nopp140-associated ribosomopathy. A null dao-5 mutant ok542 with a semi-infertile phenotype showed a delay in gonadogenesis, as well as a higher incidence of germline apoptosis. These phenotypes in dao-5(ok542) are likely resulted from inefficient rDNA transcription that was observed by run-on analyses and chromatin immunoprecipitation (ChIP) assays measuring the RNA Pol I occupancy on the rDNA promoter. ChIP assays further showed that the modifications of acetylated histone 4 (H4Ac) and dimethylation at the lysine 9 of histone 3 (H3K9me2) around the rDNA promoter were altered in dao-5 mutants compared with the N2 wild type. In addition, activated CEP-1 (a C. elegans p53 homolog) activity was also linked to the loss of DAO-5 in terms of the transcriptional upregulation of two CEP-1 downstream effectors, EGL-1 and CED-13. We propose that the dao-5 mutant of C. elegans can be a valuable model for studying human Nopp140-associated ribosomopathy at the cellular and molecular levels.
... Ribosome synthesis is an essential process that influences cell growth and development in eukaryotes (Voutev et al., 2006). Maturation of ribosomes involves a series of events including synthesis and processing of precursor rRNA, transportation of rRNA and assembly of small and large ribosomal subunits. ...
... Although some genes are involved in different steps of ribosomal maturation in various organisms, the disruption phenotypes can sometimes be quite similar. Downregulation of the ribosome biogenesis-related gene Pro1 in Caenorhabditis elegans caused slow growth, and high efficiency of RNAi could exacerbate this influence and lead to a more severe phenotype Hubbard, 2004, 2005;Voutev et al., 2006). In Aspergillus nidulans, loss of pre-rRNA processing gene CgrA resulted in slow growth and increased temperature sensitivity at the elevated temperature (Bhabhra et al., 2004). ...
Mrd1 is one of the trans-acting proteins and plays an important role in precursor ribosomal RNA processing. Here we characterized the Mamrd1 gene from Metarhizium acridum and studied its function in growth, conidiation and virulence using RNA interference. The Mamrd1 gene was identified as participating in the processing of pre-rRNA in M. acridum and was highly upregulated during the infection process. A Mamrd1-RNAi strain exhibited an appearance of fluffy mycelia, a defective branching pattern and delayed conidiation compared to the wild-type strain. Downregulation of Mamrd1 in M. acridum suppressed growth both on artificial medium and inside the insect, and significantly reduced hyphal biomass, conidium production and virulence against Locusta migratoria manilensis. These results demonstrated that Mamrd1 plays an important role in growth, conidiation and virulence in M. acridum.
... lin‐ 35 also functions non‐redundantly to represses the expression of germline‐associated genes in somatic cells and lin‐35 mutants show hypersensitivity to RNAi (Wang et al., 2005). Finally, lin‐35 negatively regulates ribosome biogenesis at the level of rRNA expression (Voutev et al., 2006). ...
... Nevertheless, our studies strongly imply that the Pro phenotype of lin‐35; spr‐1; lin‐12 (RNAi) and lin‐35; spr‐1; hop‐1(RNAi) triple mutants results from a delay in the timing of initial meiosis, consistent with previous studies (Pepper et al., 2003a;Killian and Hubbard, 2004;Killian and Hubbard, 2005). Curiously, loss of lin‐35 can also suppress the Pro phenotype of pro‐1 mutants by partially alleviating defects due to rRNA processing deficiencies (Voutev, et al., 2006). Thus, depending on developmental context and strain background, lin‐35 can act as either a suppressor or enhancer of proximal germline proliferation. ...
Using a genetic screen to identify genes that carry out redundant functions during development with lin-35/Rb, the C. elegans Retinoblastoma family ortholog, we have identified a mutation in spr-1. spr-1 encodes the C. elegans ortholog of human CoREST, a protein containing Myb-like SANT and ELM2 domains, which functions as part of a transcriptional regulatory complex. CoREST recruits mediators of transcriptional repression, including histone deacetylase, and demethylase, and interacts with the tumor suppression protein REST. spr-1/CoREST was previously shown in C. elegans to suppress defects associated with loss of the presenilin sel-12, which functions in the proteolytic processing of LIN-12/Notch. Here we show that lin-35 and spr-1 coordinately regulate several developmental processes in C. elegans including the ingression of vulval cells as well as germline proliferation. We also show that loss of lin-35 and spr-1 hypersensitizes animals to a reduction in LIN-12/Notch activity, leading to the generation of proximal germline tumors. This defect, which is observed in lin-35; spr-1; lin-12(RNAi) and lin-35; spr-1; hop-1(RNAi) triple mutants is likely due to a delay in the entry of germ cells into meiosis.
... Table 1). It is well known that ribosome biosynthesis is critical for germline stem cell differentiation, propagation and maturation of germ cells, and its perturbation can affect gonadogenesis, and also result in postembryonic growth arrest (23)(24)(25). Notably, XRN-2 has been implicated in the biogenesis and surveillance of ribosomal RNAs (rRNAs), where it facilitates the formation of mature 5.8s and 28s rRNAs by removing precursor sequences (26)(27)(28)(29). Therefore, we decided to check the status of the different rRNA species in those continuously growing worms mutant for the endoribonuclease activity of XRN-2 at the non-permissive temperature (Fig. 5A). ...
microRNAs (miRNAs) are known to regulate a vast majority of the eukaryotic genes by post-transcriptional means, and multiple nucleases play critical roles in the biogenesis and turnover of these regulators. A number of studies have indicated that turnover is important for determining the abundance of miRNAs, and thus, in turn govern their functionality. Recent research in Caenorhabditis elegans has revealed an ATP-independent endoribonuclease activity of the miRNase-XRN-2. Here, we report the characterization of this new enzymatic activity of the fundamentally important XRN-2, and show that it is critical for miRNA turnover and survival of quiescent dauer worms. The dual enzymatic activity of XRN-2 capacitates the mechanism of miRNA turnover to be dynamic, which might confer adaptive advantage to the organism. In continuously growing worms, this new enzymatic activity does not act on miRNAs, but it is important for the generation of mature ribosomal RNAs, which in turn is critical for translation, and thus indispensable for the survival of worms.
... Recently, NIR has also been shown to be necessary for asymmetric cell division in mice, suggesting a requirement in this process for correct mitotic spindle orientation [12]. In Caenorhabditis elegans, loss of function of the NOC2 homolog causes defects in gonadogenesis [13], whereas in A. thaliana, Prunet et al. showed that one of the two NOC2 homologs present, REBELOTE (RBL), is involved in floral meristem termination [14]. ...
The nucleoplasm and nucleolus are the two main territories of the nucleus. While specific functions are associated with each of these territories (such as mRNA synthesis in the nucleoplasm and ribosomal rRNA synthesis in the nucleolus), some proteins are known to be located in both. Here, we investigated the molecular function of REBELOTE (RBL), an Arabidopsis thaliana protein previously characterized as a regulator of floral meristem termination. We show that RBL displays a dual localization, in the nucleolus and nucleoplasm. Moreover, we used direct and global approaches to demonstrate that RBL interacts with nucleic acid-binding proteins. It binds to the NOC proteins SWA2, AtNOC2 and AtNOC3 in both the nucleolus and nucleoplasm, and also to OBE1 and VFP3/ENAP1. Taking into account the identities of these RBL interactors, we hypothesize that RBL acts both in ribosomal biogenesis and in the regulation of gene expression.
... We further analyzed 24 of the 40 genes: 23 that displayed more strongly elevated penetrance of the "loss of GSCs" phenotype ( Figure 5, Table 1), plus eif-6 that was previously found to cause a progenitor zone defect (Voutev et al. 2006). ...
The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In Caenorhabditis elegans, GLP-1/Notch activity prevents germline stem cell (GSC) differentiation. Other signaling mechanisms also influence the maintenance of GSCs, including the highly-conserved TOR substrate ribosomal protein S6 kinase. Although C. elegans bearing either a null mutation in rsks-1/S6K or a reduction-of-function (rf) mutation in glp-1/Notch produce half the normal number of adult germline progenitors, virtually all these single mutant animals are fertile. However, glp-1(rf)rsks-1(null) double mutant animals are all sterile, and in about half of their gonads, all GSCs differentiate, a distinctive phenotype associated with a significant reduction or loss of GLP-1 signaling. How rsks-1/S6K promotes GSC fate is unknown. Here, we determine that rsks-1/S6K acts germline-autonomously to maintain GSCs, and that it does not act through Cyclin-E or MAP kinase in this role. We found that interfering with translation also enhances glp-1(rf), but that regulation through rsks-1 cannot fully account for this effect. In a genome-scale RNAi screen for genes that act similarly to rsks-1/S6K, we identified 56 RNAi enhancers of glp-1/Notch sterility, many of which were previously not known to interact functionally with Notch. Further investigation revealed six candidates that, by genetic criteria, act linearly with rsks-1/S6K. These include genes encoding translation-related proteins, cacn-1/Cactin, an RNA exosome component and a Hedgehog-related ligand. We found that additional Hedgehog-related ligands may share functional relationships with glp-1/Notch and rsks-1/S6K in maintaining germline progenitors.
... Recently, NIR has also been shown to be necessary for asymmetric cell division in mice, suggesting a requirement in this process for correct mitotic spindle orientation [12]. In Caenorhabditis elegans, loss of function of the NOC2 homolog causes defects in gonadogenesis [13], whereas in A. thaliana, Prunet et al. showed that one of the two NOC2 homologs present, REBELOTE (RBL), is involved in floral meristem termination [14]. ...
The nucleoplasm and nucleolus are the two main territories of the nucleus. While specific functions are associated with each of these territories (such as mRNA synthesis in the nucleoplasm and ribosomal rRNA synthesis in the nucleolus), some proteins are known to be located in both. Here, we investigated the molecular function of REBELOTE (RBL), an Arabidopsis thaliana protein previously characterized as a regulator of floral meristem termination. We show that RBL displays a dual localization, in the nucleolus and nucleoplasm. Moreover, we used direct and global approaches to demonstrate that RBL interacts with nucleic acid‐binding proteins. It binds to the NOC proteins SWA2, AtNOC2 and AtNOC3 in both the nucleolus and nucleoplasm, and also to OBE1 and VFP3/ENAP1. Taking into account the identities of these RBL interactors, we hypothesise that RBL acts both in ribosomal biogenesis and in the regulation of gene expression.
... The most common abnormalities fell on a spectrum ranging from gonads enlarged with proliferative germ cell nuclei (detected as Pmex-5::H2B::mCherry-positive, "mex-5 + ") to uterine masses consisting of largely Pmex-5::H2B::mCherry-negative cells ("mex-5 -", Fig. 1C). The mex-5 + gonad enlargements resembled proximal germ cell tumors similar to those caused by "latent niche" signaling (5, 6), and the mex-15 5uterine masses were disorganized and appeared to contain differentiated cells. Indeed, the majority of uterine masses expressed elt-1 and unc-119 reporter genes, embryonic markers of epidermis and neurons, confirming somatic differentiation ( Fig. 1D and E). ...
The roundworm C. elegans transiently arrests larval development to survive extended starvation ( 1 ), but such early-life starvation reduces reproductive success ( 2, 3 ). Maternal dietary restriction (DR) buffers progeny from starvation, increasing reproductive success ( 4 ). It is unknown why early-life starvation decreases reproductive success and how maternal diet modifies this process. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of abnormalities in the reproductive system, including glp-1/ Notch-sensitive germ-cell tumors and uterine masses that express neuronal and epidermal markers. We found that maternal DR reduces the penetrance of starvation-induced abnormalities, including tumors. Furthermore, we show that maternal DR reduces insulin/IGF signaling (IIS) in progeny, and that daf-16 /FoxO and skn-1 /Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR to suppress abnormalities. daf-16 /FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals complex inter- and intra-generational effects of nutrient availability mediated by IIS with consequences on developmental integrity and reproductive success.
One Sentence Summary
Intergenerational effects of diet on IIS
... Following your nice suggestion, we used northern blotting to analyze pre-rRNA level and rRNA processing in WT and rrp-8(kun54) worms. We designed three probes to detect pre-rRNA level and rRNA intermediates (b, b', c and c') according to previous reports ( Figure 2E) (Bousquet-Antonelli et al., 2000;Saijou et al., 2004;Voutev et al., 2006). Digoxin-labeled RNA probes were generated using DIG northern starter kit (Roche). ...
... T04A8.6, K01C8.9, and rbd-1 [69]; dao-5 [70]; and nol-6 [71]. The concomitant depletion of multiple UUGUU-bearing transcripts and accumulation of NCL-1 protein in oocytes raises the possibility that NCL-1 promotes dissolution of the nucleolus in the -1 oocyte, at least in part, by coordinated translational repression, deadenylation, and/or degradation of transcripts involved in nucleolar biogenesis. ...
Background:
The 3' untranslated regions (UTRs) of mRNAs play a major role in post-transcriptional regulation of gene expression. Selection of transcript cleavage and polyadenylation sites is a dynamic process that produces multiple transcript isoforms for the same gene within and across different cell types. Using LITE-Seq, a new quantitative method to capture transcript 3' ends expressed in vivo, we have characterized sex- and cell type-specific transcriptome-wide changes in gene expression and 3'UTR diversity in Caenorhabditis elegans germline cells undergoing proliferation and differentiation.
Results:
We show that nearly half of germline transcripts are alternatively polyadenylated, that differential regulation of endogenous 3'UTR variants is common, and that alternative isoforms direct distinct spatiotemporal protein expression patterns in vivo. Dynamic expression profiling also reveals temporal regulation of X-linked gene expression, selective stabilization of transcripts, and strong evidence for a novel developmental program that promotes nucleolar dissolution in oocytes. We show that the RNA-binding protein NCL-1/Brat is a posttranscriptional regulator of numerous ribosome-related transcripts that acts through specific U-rich binding motifs to down-regulate mRNAs encoding ribosomal protein subunits, rRNA processing factors, and tRNA synthetases.
Conclusions:
These results highlight the pervasive nature and functional potential of patterned gene and isoform expression during early animal development.
... In eukaryotic system the ribosome biogenesis is an essential metabolic activity connected with pivotal cellular processes like cell proliferation and differentiation [81]. Especially in invertebrate species like C. elegans the ribosomal biogenesis regulates gonadogenesis by assisting in germline proliferation and pattern formation [82]. The transcriptome annotation details of earthworm E. fetida have represented several important translational regulatory factors like 60S ribosomal L5, 60S ribosomal L3, 40S ribosomal S13, 40S ribosomal S5 and 40S ribosomal S20, which may serve as the regulatory factors to assist in the biogenesis and structural constitution of ribosome in the worm species. ...
In annelid worms, the nerve cord serves as a crucial organ to control the sensory and behavioral physiology. The inadequate genome resource of earthworms has prioritized the comprehensive analysis of their transcriptome dataset to monitor the genes express in the nerve cord and predict their role in the neurotransmission and sensory perception of the species. The present study focuses on identifying the potential transcripts and predicting their functional features by annotating the transcriptome dataset of nerve cord tissues prepared by Gong et al., 2010 from the earthworm Eisenia fetida. Totally 9762 transcripts were successfully annotated against the NCBI nr database using the BLASTX algorithm and among them 7680 transcripts were assigned to a total of 44,354 GO terms. The conserve domain analysis indicated the over representation of P-loop NTPase domain and calcium binding EF-hand domain. The COG functional annotation classified 5860 transcript sequences into 25 functional categories. Further, 4502 contig sequences were found to map with 124 KEGG pathways. The annotated contig dataset exhibited 22 crucial neuropeptides having considerable matches to the marine annelid Platynereis dumerilii, suggesting their possible role in neurotransmission and neuromodulation. In addition, 108 human stem cell marker homologs were identified including the crucial epigenetic regulators, transcriptional repressors and cell cycle regulators, which may contribute to the neuronal and segmental regeneration. The complete functional annotation of this nerve cord transcriptome can be further utilized to interpret genetic and molecular mechanisms associated with neuronal development, nervous system regeneration and nerve cord function.
... SAGE analysis has indicated that these genes are expressed in dissected gonads, though multiple somatic gonad cells including the DTC are present in this sample (Wang et al., 2009). Notably, pab-1, W07E6.2, and par-5 have reported roles in germ cell proliferation (Aristizábal-Corrales et al., 2012;Ko et al., 2010;Voutev et al., 2006). Germline-expression profiling previously showed that his-72, ran-1, W07E6.2, and snr-2 are enriched in wild-type animals compared to germline-depleted mutants (Reinke et al., 2004), suggesting that these genes are expressed in the germline. ...
Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.
... Among genes with low hermaphroditebiased expression, endocytosis function was enriched. These genes included pro-2, known to regulate germline growth and development (Voutev et al. 2006), and mrps-18B, which plays a role in vulva formation (Ceron et al. 2007). Among genes with low male-biased expression, lysosome-associated genes were enriched, including rab-7, a Rab GTPase required for lysosome trafficking. ...
Dosage compensation mechanisms equalize the level of X chromosome expression between sexes. Yet the X chromosome is often enriched for genes exhibiting sex-biased, i.e. imbalanced expression. The relationship between X chromosome dosage compensation and sex-biased gene expression remains largely unexplored. Most studies determine sex biased gene expression without distinguishing between contributions from X chromosome copy number (dose) and the animal's sex. Here, we uncoupled X chromosome dose from sex-specific gene regulation in C. elegans to determine the effect of each on X expression. In early embryogenesis, when dosage compensation is not yet fully active, X chromosome dose drives the hermaphrodite-biased expression of many X-linked genes, including several genes that were shown to be responsible for hermaphrodite fate. A similar effect is seen in the C. elegans germline, where X chromosome dose contributes to higher hermaphrodite X expression, suggesting that lack of dosage compensation in the germline may have a role in supporting higher expression of X chromosomal genes with female-biased functions in the gonad. In the soma, dosage compensation effectively balances X expression between the sexes. As a result, somatic sex-biased expression is almost entirely due to sex-specific gene regulation. These results suggest that lack of dosage compensation in different tissues and developmental stages allow X chromosome copy number to contribute to sex-biased gene expression and function.
... The distal sheath cells (Sh1-2) promote germ line proliferation and exit from meiotic pachytene (Killian and Hubbard, 2005;McCarter et al., 1997), whereas the 3 pairs of proximal sheath cells (Sh 3-5) are important for oocyte maturation and for the smooth-muscle-like contractions that push oocytes into the spermatheca for fertilization (Strome, 1986;Miller et al., 2003Miller et al., , 2001McCarter et al., 1999). Defects in sheath cells, including mutations in sheath cell expressed genes like ceh-18, a POU class homeoprotein (Rose et al., 1997), pro-1, a protein involved in rRNA processing (Voutev et al., 2006;Killian and Hubbard, 2004), and mup-2, a troponin homolog (Myers et al., 1996), or ablation of sheath cell precursor cells (Sh1-5) (Killian and Hubbard, 2005;McCarter et al., 1997) produce an array of germ line phenotypes including tumors (Killian and Hubbard, 2005;McCarter et al., 1997;Killian and Hubbard, 2004), sperm filled proximal gonad arms that lack oocytes (Killian and Hubbard, 2005) and endo-mitotically duplicating oocytes (Killian and Hubbard, 2005;McCarter et al., 1997;Rose et al., 1997;Myers et al., 1996) (the Emo phenotype (Iwasaki et al., 1996)). Even though it is clear that sheath cell development has a crucial role in proper germ line formation, little is known about the molecular mechanisms that control sheath cell specification, development and patterning in C. elegans. ...
CACN-1/Cactin is a conserved protein identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode Caenorhabditis elegans. In addition to possessing distal tip cells that migrate past their correct stopping point, animals depleted of cacn-1 are sterile. In this study, we show that CACN-1 is needed in the soma for proper germ line development and maturation. When CACN-1 is depleted, sheath cells are absent and/or abnormal. When sheath cells are absent, hermaphrodites produce sperm, but do not switch appropriately to oocyte production. When sheath cells are abnormal, some oocytes develop but are not successfully ovulated and undergo endomitotic reduplication (Emo). Our previous proteomic studies show that CACN-1 interacts with a network of splicing factors. Here, these interactors were screened for germ line phenotypes using RNAi. Depletion of many of these factors led to missing or abnormal sheath cells and germ line defects, particularly absent and/or Emo oocytes. These results suggest CACN-1 is part of a protein network that influences somatic gonad development and function through alternative splicing or post-transcriptional gene regulation.
... Our previous results Hubbard, 2004, 2005;Korta et al., 2012;Voutev et al., 2006) and those of others (Ariz et al., 2009;Beanan and Strome, 1992;Fox et al., 2011) indicate that signals promoting the proliferative cell fate can be separated from those that promote robust mitotic cell cycle progression during larval stages. For example, GLP-1/ Notch signaling maintains the undifferentiated proliferative-competent fate of germ cells and/or inhibits meiotic entry (Hansen and Schedl, 2013). ...
Many organisms accumulate a pool of germline stem cells during development that is maintained in later life. The dynamics of establishment, expansion and homeostatic maintenance of this pool are subject to both developmental and physiological influences including the availability of a suitable niche microenvironment, nutritional status, and age. Here, we investigated the dynamics of germline proliferation during stages of expansion and homeostasis, using the C. elegans germ line as a model. The vast majority of germ cells in the proliferative zone are in interphase stages of mitosis (G1, S, G2) rather than in the active mitotic (M) phase. We examined mitotic index and DNA content, comparing different life stages, mutants, and physiological conditions. We found that germ cells in larval stages cycle faster than in adult stages, but that this difference could not be attributed to sexual fate of the germ cells. We also found that larval germ cells exhibit a lower average DNA content compared to adult germ cells. We extended our analysis to consider the effects of distance from the niche and further found that the spatial pattern of DNA content differs between larval and adult stages in the wild type and among mutants in pathways that interfere with cell cycle progression, cell fate, or both. Finally, we characterized expansion of the proliferative pool of germ cells during adulthood, using a regeneration paradigm (ARD recovery) in which animals are starved and re-fed. We compared adult stage regeneration and larval stage expansion, and found that the adult germ line is capable of rapid accumulation but does not sustain a larval-level mitotic index nor does it recapitulate the larval pattern of DNA content. The regenerated germ line does not reach the number of proliferative zone nuclei seen in the continuously fed adult. Taken together, our results suggest that cell cycle dynamics are under multiple influences including distance from the niche, age and/or maturation of the germ line, nutrition and, possibly, latitude for physical expansion.
... DNA probes were generated from PCR products amplified from C. elegans genomic DNA and labeled with 32 P-dCTP (Perkin Elmer, PK-BLU513H) by hexamer priming. Primers for generating the ribosomal RNA species and actin probes were performed as described by Voutev et al. [46]. Hybridization was carried out at 55°C in 0.36 M Na 2 HPO 4 , 0.14 M NaH 2 PO 4 , 1 mM EDTA, 10% SDS, 25% formamide and 0.1 mg/ml salmon sperm DNA. ...
Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3'UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.
... IPI3, the RID3 ortholog of budding yeast is a component of a pre-rRNA processing complex, and the IPI3 shutoff experiment has revealed its function in pre-rRNA processing (Krogan et al., 2004). PRO-1, the RID3 ortholog of C. elegans, has also been reported to mediate pre-rRNA processing (Voutev et al., 2006). On the basis of temperature-dependent accumulation of rRNA precursors in rid3, we showed that RID3 was involved in pre-rRNA processing. ...
At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.
... It is not clear why loss of mel-28 function enhances the reduced brood size defects caused by genes involved in translation initiation. However, there are previously established links between gonadogenesis and ribosome biogenesis in C. elegans (Voutev et al. 2006;Kudron and Reinke 2008). n Table 3 Genetic interaction tests with mel-28 and nuclear pore complex components Proteasome: Y39C12A.1 encodes a homolog of Nas1p, a subunit of the 26S proteasome (Hori et al. 1998). ...
mel-28 (maternal-effect-lethal-28) encodes a conserved protein required for nuclear envelope function and chromosome segregation in Caenorhabditis elegans. As mel-28 is a strict maternal-effect lethal gene, its function is required in the early embryo but appears to be dispensable for larval development. We wanted to test the idea that mel-28 has postembryonic roles that are buffered by the contributions of other genes. To find genes that act coordinately with mel-28, we did an RNAi-based genetic interaction screen using mel-28 and wild-type larvae. We screened 18,364 clones and identified 65 genes that cause sterility in mel-28 but not wild-type worms. Some of these genes encode components of the nuclear pore. In addition we identified genes involved in dynein and dynactin function, vesicle transport, and cell-matrix attachments. By screening mel-28 larvae we have bypassed the requirement for mel-28 in the embryo, uncovering pleiotropic functions for mel-28 later in development that are normally provided by other genes. This work contributes toward revealing the gene networks that underlie cellular processes and reveals roles for a maternal-effect lethal gene later in development.
... Implication of Nle in ribosome biogenesis has not been directly addressed so far in other eukaryotes. Nonetheless, Solanum chacoense NLE and MDN1 were found to interact in yeast two-hybrid assay (Chantha and Matton, 2007), and similar phenotypes were obtained after knockdown of Nle and orthologues of yeast genes implicated in ribosome biogenesis in Arabidopsis thaliana and Caenorhabditis elegans (Voutev et al., 2006;Chantha et al., 2010). In the mouse, we previously reported that constitutive Nle loss of function results in early embryonic lethality and that Nle is mainly required in inner cell mass cells, being instrumental for their survival (Cormier et al., 2006). ...
Blood cell production relies on the coordinated activities of hematopoietic stem cells (HSCs) and multipotent and lineage-restricted progenitors. Here, we identify Notchless (Nle) as a critical factor for HSC maintenance under both homeostatic and cytopenic conditions. Nle deficiency leads to a rapid and drastic exhaustion of HSCs and immature progenitors and failure to maintain quiescence in HSCs. In contrast, Nle is dispensable for cycling-restricted progenitors and differentiated cells. In yeast, Nle/Rsa4 is essential for ribosome biogenesis, and we show that its role in pre-60S subunit maturation has been conserved in the mouse. Despite its implication in this basal cellular process, Nle deletion affects ribosome biogenesis only in HSCs and immature progenitors. Ribosome biogenesis defects are accompanied by p53 activation, which causes their rapid exhaustion. Collectively, our findings establish an essential role for Nle in HSC and immature progenitor functions and uncover previously unsuspected differences in ribosome biogenesis that distinguish stem cells from restricted progenitor populations.
... Implication of Nle in ribosome biogenesis has not been directly addressed so far in other eukaryotes. Nonetheless, Solanum chacoense NLE and MDN1 were found to interact in yeast two-hybrid assay (Chantha and Matton, 2007), and similar phenotypes were obtained after knockdown of Nle and orthologues of yeast genes implicated in ribosome biogenesis in Arabidopsis thaliana and Caenorhabditis elegans (Voutev et al., 2006;Chantha et al., 2010). In the mouse, we previously reported that constitutive Nle loss of function results in early embryonic lethality and that Nle is mainly required in inner cell mass cells, being instrumental for their survival (Cormier et al., 2006). ...
... Pro tumors are often very robust, quickly fi lling the entire proximal end of the gonad with proliferative cells. While there appear to be diverse molecular and physiological origins for proximal tumor formation, most, if not all, seem to result in inappropriate contact between undifferentiated proximal germ cells and gonadal sheath cells (Killian and Hubbard 2004 ;Pepper et al. 2003 ;Seydoux et al. 1990 ;Voutev et al. 2006 ) . During normal larval development, there is a period when all germ cells are proliferative, prior to when some proximal cells enter meiotic prophase. ...
The C. elegans germ line has emerged as an important model for -understanding how a stem cell population is maintained throughout the life of the animal while still producing the gametes necessary for propagation of the species. The stem cell population in the adult hermaphrodite is relatively large, with stem cells giving rise to daughters that appear intrinsically equivalent; however, some of the daughters retain the proliferative fate while others enter meiotic prophase. While machinery exists for cells to progress through the mitotic cell cycle and machinery exists for cells to progress through meiotic prophase, central to understanding germ line development is identifying the genes and regulatory processes that determine whether the mitotic cell cycle or meiotic prophase machinery will be utilized; in other words, the genes that regulate the switch of germ cells from the proliferative stem cell fate to the meiotic development fate. Whether a germ cell self-renews or enters meiotic prophase is largely determined by its proximity to the distal tip cell (DTC), which is the somatic niche cell that caps the distal end of the gonad. Germ cells close to the DTC have high levels of GLP-1 Notch signaling, which promotes the proliferative fate, while cells further from the DTC have high activity levels of the GLD-1 and GLD-2 redundant RNA regulatory pathways, as well as a third uncharacterized pathway, each of which direct cells to enter meiotic prophase. Other factors and pathways modulate this core genetic pathway, or work in parallel to it, presumably to ensure that a tight balance is maintained between proliferation and meiotic entry.
... Killian and Hubbard reported the C. elegans " proximal tumor " phenotype , exhibited by the hermaphrodite gonad, this was the first phenotype associated with loss of nucleolar integrity and impaired ribosome biogenesis function [63]. Hubbard mapped this unusual phenotype to pro genes (pro-1, pro2, and pro-3), which encode factors involved in ribosome biogenesis [63, 64]. RNAi screening against other factors involved in ribosome biogenesis revealed a similar gonadogenesis phenotype, suggesting that such phenotypes can result from inefficient ribosome biogenesis. ...
Nucleolar size and appearance correlate with ribosome biogenesis and cellular activity. The mechanisms underlying changes in nucleolar appearance and regulation of nucleolar size that occur during differentiation and cell cycle progression are not well understood. Caenorhabditis elegans provides a good model for studying these processes because of its small size and transparent body, well-characterized cell types and lineages, and because its cells display various sizes of nucleoli. This paper details the advantages of using C. elegans to investigate features of the nucleolus during the organism's development by following dynamic changes in fibrillarin (FIB-1) in the cells of early embryos and aged worms. This paper also illustrates the involvement of the ncl-1 gene and other possible candidate genes in nucleolar-size control. Lastly, we summarize the ribosomal proteins involved in life span and innate immunity, and those homologous genes that correspond to human disorders of ribosomopathy.
... Our literature search revealed that close to 50 studies used the rrf-1(pk1417) mutant to investigate the role of a gene of interest in the germline versus somatic tissues. However, several of these publications used additional and alternative methods to confirm a germline requirement for the gene of interest, such as mosaic analysis [31,32,33,34,35]. Indeed, most of the published studies used rrf-1(pk1417) mutants to determine if a gene expressed in the gonad has a specific role in the germline or the somatic gonad [36,37,38]. ...
Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene sod-3; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools.
... We discovered genes that are required for robust larval germline proliferation but that do not act in the DTC by analysis of mutants from genetic screens that identified "pro" genes (Killian and Hubbard, 2004;Voutev et al., 2006). For pro-1, the activity was pinpointed to the sheath lineage of the somatic gonad ( Fig. 3.1). ...
Germline proliferation in Caenorhabditis elegans is emerging as a compelling model system for understanding the molecular basis for the developmental and physiological control of cell proliferation. This review covers the discovery and implications of the role of the insulin/IGF-like signaling pathway in germline proliferation during germline development. This pathway plays a host of important roles in C. elegans biology. Its role in germline proliferation is important to generate the proper adult stem/progenitor population and to ensure optimal fecundity. Moreover, in this role, it is restricted to reproductive (as opposed to dauer) larval stages and impinges on the G2 of the cell cycle. Two putative insulin ligands are especially important for the germline role but do not mediate signaling in other tissues. A picture is emerging of a complex web of developmentally and temporally restricted, ligand- and tissue-specific responses to insulin signaling. Avenues for future studies include the regulation of specific insulin-like ligands and the mechanisms for tissue-specific responses to them.
... Ablation of Sh1 causes a reduced rate of GSC proliferation, indicating a role for Sh1 cells in promoting GSC proliferation (Killian and Hubbard, 2005). How do the Sh1 cells encourage proliferation of the underlying GSCs during L3 and early L4? Optimal ribosome biogenesis within the Sh1 cells is likely to be especially important for the signals that promote proliferation of GSCs during L3 and L4 ( Killian and Hubbard, 2004;Voutev et al., 2006). Ribosome biogenesis is also crucial within the GSCs themselves, as mutations that affect rRNA levels have drastic effects upon GSC proliferation during larval development ( Kudron and Reinke, 2008 The signals that promote the proliferation of GSCs originate adjacent to the MZ of the germ line, limiting proliferation to this region and permitting differentiation and gametogen-esis in the proximal arms of gonads. ...
The germ cells of Caenorhabditis elegans serve as a useful model to study the balance between proliferation and differentiation within the context of development and changing environmental signals experienced by the animal. Germ cells adjacent to a stem cell niche in the distal region of the gonad retain the capacity to divide during adulthood, making them unique from other cells in the organism. We will highlight recent advances in our understanding of mechanisms that control proliferation, as well as the signaling pathways involved in promoting mitosis at the expense of differentiation.
... The ring canals allow the transfer of cytoplasm from the nurse cells to the oocyte, a key process for egg maturation. In C. elegans, the sdad1 gene is required for normal gonadal development and regulation of the germline proliferation and differentiation (Voutev et al., 2006). The preferential expression of the genes in oocytes, together with the data gained from non-vertebrate orthologs strongly, suggests that the nup54, klhl8, and sdad1 genes may have a role in fish oogenesis. ...
The gonadal soma-derived factor (GSDF) belongs to the transforming growth factor-β superfamily and is conserved in teleostean fish species. Gsdf is specifically expressed in the gonads, and gene expression is restricted to the granulosa and Sertoli cells in trout and medaka. The gsdf gene expression is correlated to early testis differentiation in medaka and was shown to stimulate primordial germ cell and spermatogonia proliferation in trout. In the present study, we show that the gsdf gene localizes to a syntenic chromosomal fragment conserved among vertebrates although no gsdf-related gene is detected on the corresponding genomic region in tetrapods. We demonstrate using quantitative RT-PCR that most of the genes localized in the synteny are specifically expressed in medaka gonads. Gsdf is the only gene of the synteny with a much higher expression in the testis compared to the ovary. In contrast, gene expression pattern analysis of the gsdf surrounding genes (nup54, aff1, klhl8, sdad1, and ptpn13) indicates that these genes are preferentially expressed in the female gonads. The tissue distribution of these genes is highly similar in medaka and zebrafish, two teleostean species that have diverged more than 110 million years ago. The cellular localization of these genes was determined in medaka gonads using the whole-mount in situ hybridization technique. We confirm that gsdf gene expression is restricted to Sertoli and granulosa cells in contact with the premeiotic and meiotic cells. The nup54 gene is expressed in spermatocytes and previtellogenic oocytes. Transcripts corresponding to the ovary-specific genes (aff1, klhl8, and sdad1) are detected only in previtellogenic oocytes. No expression was detected in the gonocytes in 10 dpf embryos. In conclusion, we show that the gsdf gene localizes to a syntenic chromosomal fragment harboring evolutionary conserved genes in vertebrates. These genes are preferentially expressed in previtelloogenic oocytes, and thus, they display a different cellular localization compared to that of the gsdf gene indicating that the later gene is not co-regulated. Interestingly, our study identifies new clustered genes that are specifically expressed in previtellogenic oocytes (nup54, aff1, klhl8, sdad1).
... Perhaps because of their high expression and fundamental importance to cell viability, the vast majority of factors that displayed phenotypes in the RNAi screen encode proteins implicated in ribosome biogenesis. Previous studies have linked the mutation of specific regulators of ribosome biogenesis to defective germ cell proliferation (Killian and Hubbard, 2004; Kudron and Reinke, 2008; Voutev et al., 2006), suggesting that both the somatic gonad and the germ line are especially sensitive to changes in ribosome production. In support of this possibility, microarray experiments directly comparing mitotic and meiotic germ cell populations in dissected distal gonad tips identified genes encoding factors regulating translation, including essentially all ribosome components, as strongly enriched in the mitotic germ cells (W. ...
Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.
... Our results show that disruption of ribosomes via treatment with actinomycin D or by RNAi-mediated knockdown of individual ribosomal protein subunit (rps) genes leads to an enhanced resistance to S. enterica infection. Ribosomal proteins are required for proper germline development [65,66] and CEP-1 plays a role in stressinduced germline apoptosis in C. elegans [67]. To address whether the germline may affect the enhanced resistance to pathogen infection of nol-6 or rps RNAi nematodes, RNAi was performed in germline-deficient animals glp-4(bn2). ...
Author Summary
Innate immunity comprises a variety of defense mechanisms used by metazoans to prevent microbial infections. These nonspecific defense responses used by the innate immune system are governed by interacting and intersecting pathways that control not only immune responses but also longevity and responses to different stresses. Increasing evidence highlights the plurifunctional nature of the nucleolus, which appears to control various cellular processes involved in health and disease, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. We provide evidence indicating that the nucleolus suppresses innate immunity against bacteria by preventing the transcriptional activity of the tumor suppressor p53. We found that animals lacking nucleolar proteins are highly resistant to infections by bacterial pathogens. We also found that the activation of innate immunity by inhibition of nucleolar proteins requires potential immune effectors whose expression in response to stress is regulated by p53. Our study links the nucleolus, p53, and innate immunity against bacterial infections for the first time, and highlights a new mechanism that can potentially be exploited to alleviate bacterial infections.
... ( J) Quantification of A-H. Bar, 20 mm. and White 1981;McCarter et al. 1997;Killian and Hubbard 2004;Voutev et al. 2006), we used mosaic analysis to determine whether METT-10 acts in the soma or the germ line to inhibit germ cell proliferative fate. We generated mosaics by taking advantage of the fact that extrachromosomal arrays are imperfectly transmitted during cell division (Yochem and Herman 2005). ...
Germ-line stem cells are unique because they either self-renew through mitosis or, at a certain frequency, switch to meiosis and produce gametes. The switch from proliferation to meiosis is tightly regulated, and aberrations in switching result in either too little or too much proliferation. To understand the genetic basis of this regulation, we characterized loss-of-function mutations and a novel tumorous allele of Caenorhabditis elegans mett-10, which encodes a conserved putative methyltransferase. We show that METT-10 is a nuclear protein that acts in the germ line to inhibit the specification of germ-cell proliferative fate. METT-10 also promotes vulva, somatic gonad, and embryo development and ensures meiotic development of those germ cells that do differentiate. In addition, phenotypic analysis of a mett-10 null allele reveals that METT-10 enables mitotic cell cycle progression. The finding that METT-10 functions to inhibit germ-cell proliferative fate, despite promoting mitotic cell cycle progression of those germ cells that do proliferate, separates the specification of proliferative fate from its execution.
... This and other work has led to the identification of a diverse array of redundant functions for LIN-35 including roles in cell cycle control [14,15], cell fate specification [16], asymmetric cell division [17], larval growth [18,19], fertility [16,20], organogenesis [20,21], and organ function [22]. In addition, LIN-35, along with a number of other class B SynMuv genes, has been shown to function nonredundantly in the control of transgene expression [23], RNAi [24,25], germline and somatic sex-linked apoptosis [26,27], ribosome biogenesis [28], and the somatic silencing of germline gene expression [13,25]. In our current work, we have sought to understand the mechanistic basis for the synthetic genetic interactions observed between lin-35 and two mutations previously identified by our screen, ubc-18 and pha-1 [21,29]. ...
Genetic redundancy, whereby two genes carry out seemingly overlapping functions, may in large part be attributable to the intricacy and robustness of genetic networks that control many developmental processes. We have previously described a complex set of genetic interactions underlying foregut development in the nematode Caenorhabditis elegans. Specifically, LIN-35/Rb, a tumor suppressor ortholog, in conjunction with UBC-18-ARI-1, a conserved E2/E3 complex, and PHA-1, a novel protein, coordinately regulates an early step of pharyngeal morphogenesis involving cellular re-orientation. Functional redundancy is indicated by the observation that lin-35; ubc-18 double mutants, as well as certain allelic combinations of pha-1 with either lin-35 or ubc-18, display defects in pharyngeal development, whereas single mutants do not. Using a combination of genetic and molecular analyses, we show that sup-35, a strong recessive suppressor of pha-1-associated lethality, also reverts the synthetic lethality of lin-35; ubc-18, lin-35; pha-1, and ubc-18 pha-1 double mutants. SUP-35, which contains C2H2-type Zn-finger domains as well as a conserved RMD-like motif, showed a dynamic pattern of subcellular localization during embryogenesis. We find that mutations in sup-35 specifically suppress hypomorphic alleles of pha-1 and that SUP-35, acting genetically upstream of SUP-36 and SUP-37, negatively regulates pha-1 transcription. We further demonstrate that LIN-35, a transcriptional repressor, and UBC-18-ARI-1, a complex involved in ubiquitin-mediated proteolysis, negatively regulate SUP-35 abundance through distinct mechanisms. We also show that HCF-1, a C. elegans homolog of host cell factor 1, functionally antagonizes LIN-35 in the regulation of sup-35. Our cumulative findings piece together the components of a novel regulatory network that includes LIN-35/Rb, which functions to control organ morphogenesis. Our results also shed light on general mechanisms that may underlie developmental genetic redundancies as well as principles that may govern complex disease traits.
Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in C. elegans that is marked by an abnormal globular morphology of the nucleolus and fewer germ cells. We identified DCAF-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of germline-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor PWP1. We observed that inactivation of C. elegans DCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.
Cell invasion through basement membrane (BM) barriers is important in development, immune function, and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of C. elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including TCTP (translationally controlled tumor protein). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling, and ribosome biogenesis analysis revealed a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands prior to invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.
Stem cells are tightly controlled in vivo . Both the balance between self-renewal and differentiation and the rate of proliferation are often regulated by multiple factors. The Caenorhabditis elegans hermaphrodite germ line provides a simple and accessible system for studying stem cells in vivo . In this system, GLP-1/Notch activity prevents the differentiation of distal germ cells in response to ligand production from the nearby distal tip cell, thereby supporting a stem cell pool. However, a delay in germline development relative to somatic gonad development can cause a pool of undifferentiated germ cells to persist in response to alternate Notch ligands expressed in the proximal somatic gonad. This pool of undifferentiated germ cells forms a proximal tumor that, in adulthood, blocks the oviduct. This type of "latent niche"-driven proximal tumor is highly penetrant in worms bearing the temperature-sensitive weak gain-of-function mutation glp-1(ar202) at the restrictive temperature. At the permissive temperature, few worms develop tumors. Nevertheless, several interventions elevate the penetrance of proximal tumor formation at the permissive temperature, including reduced insulin signaling or the ablation of distal-most sheath cells. To systematically identify genetic perturbations that enhance proximal tumor formation, we sought genes that, upon RNAi depletion, elevate the percentage of worms bearing proximal germline tumors in glp-1(ar202) at the permissive temperature. We identified 43 genes representing a variety of functional classes, the most enriched of which is "translation". Some of these genes also influence the distal germ line, and some are conserved genes for which genetic interactions with Notch were not previously known in this system.
Matrix metalloproteinases (MMPs) are associated with decreased patient prognosis but have failed as anti-invasive drug targets despite promoting cancer cell invasion. Through time-lapse imaging, optical highlighting, and combined genetic removal of the five MMPs expressed during anchor cell (AC) invasion in C. elegans, we find that MMPs hasten invasion by degrading basement membrane (BM). Though irregular and delayed, AC invasion persists in MMP− animals via adaptive enrichment of the Arp2/3 complex at the invasive cell membrane, which drives formation of an F-actin-rich protrusion that physically breaches and displaces BM. Using a large-scale RNAi synergistic screen and a genetically encoded ATP FRET sensor, we discover that mitochondria enrich within the protrusion and provide localized ATP that fuels F-actin network growth. Thus, without MMPs, an invasive cell can alter its BM-breaching tactics, suggesting that targeting adaptive mechanisms will be necessary to mitigate BM invasion in human pathologies.
NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-clinique.
Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to “disentangle” these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25°; for comparison, wild-type oogenic hermaphrodites were also raised at 25°. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.
Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to "disentangle" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25C; for comparison, wild-type oogenic hermaphrodites were also raised at 25C. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.
Stem cells, their niches, and their relationship to cancer are under intense investigation. Because tumors and metastases acquire self-renewing capacity, mechanisms for their establishment may involve cell-cell interactions similar to those between stem cells and stem cell niches. On the basis of our studies in Caenorhabditis elegans, we introduce the concept of a "latent niche" as a differentiated cell type that does not normally contact stem cells nor act as a niche but that can, under certain conditions, promote the ectopic self-renewal, proliferation, or survival of competent cells that it inappropriately contacts. Here, we show that ectopic germ-line stem cell proliferation in C. elegans is driven by a latent niche mechanism and that the molecular basis for this mechanism is inappropriate Notch activation. Furthermore, we show that continuous Notch signaling is required to maintain ectopic germ-line proliferation. We highlight the latent niche concept by distinguishing it from a normal stem cell niche, a premetastatic niche and an ectopic niche. One of the important distinguishing features of this mechanism for tumor initiation is that it could operate in the absence of genetic changes to the tumor cell or the tumor-promoting cell. We propose that a latent niche mechanism may underlie tumorigenesis and metastasis in humans.
Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. We cloned fog-2 by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3′untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. We propose that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species.
p27BBP/eIF6 is an evolutionarily conserved protein that was originally identified as p27BBP, an interactor of the cytoplasmic domain of integrin β4 and, independently, as the putative translation initiation factor
eIF6. To establish the in vivo function of p27BBP/eIF6, its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene
was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing β4 integrin, p27BBP/eIF6 is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of β4 integrin. Surprisingly,
in the absence and in the presence of the β4 integrin subunit, p27BBP/eIF6 is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27BBP/eIF6 homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level
of free ribosomal 60S subunit. Furthermore, human p27BBP/eIF6 can rescue the lethal effect of the iihΔ yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27BBP/eIF6 in ribosome biogenesis or assembly rather than in translation. A further function related to the β4 integrin subunit may
have evolved specifically in higher eukaryotic cells.
Regulation of ribosome synthesis is an essential aspect of growth control. Thus far, little is known about the factors that control and coordinate these processes. We show here that the Caenorhabditis elegans gene ncl-1 encodes a zinc finger protein and may be a repressor of RNA polymerase I and III transcription and an inhibitor of cell growth. Loss of function mutations in ncl-1, previously shown to result in enlarged nucleoli, result in increased rates of rRNA and 5S RNA transcription and enlarged cells. Furthermore, ncl-1 adult worms are larger, have more protein, and have twice as much rRNA as wild-type worms. Localization studies show that the level of NCL-1 protein is independently regulated in different cells of the embryo. In wild-type embryos, cells with the largest nucleoli have the lowest level of NCL-1 protein. Based on these results we propose that ncl-1 is a repressor of ribosome synthesis and cell growth.
In the nematode Caenorhabditis elegans, each of its 302 individual neurons is an identified neuron. We have screened more than 100 mutations affecting locomotion in C. elegans immunocytochemically, using monoclonal antibodies that recognize specific subsets of neurons. Mutations in 25 genes affect the axonal outgrowth and guidance of a set of 6 mechanosensory receptor neurons (ALML, ALMR, AVM, PVM, PLML and PLMR). Similarly, mutations in 14 genes alter the axonal growth and process placement of two classes of inhibitory motor neurons (DD and VD). Most of these genes affect both embryonic and postembryonic development of the C. elegans nervous system, and have variable expressivity. Our results suggest that different neuron types are specified by a combination of genes that are activated in different cell types. Molecular characterization of such genes could lead to the identification of molecules critical in axonal outgrowth and guidance in higher organisms.
Cell-cell signaling controls the specification of vulval cell fates in Caenorhabditis elegans. Although previous studies have identified genes that function at early steps in the signaling pathway, the late steps are not well understood. Here, we begin to characterize those late events by showing that the lin-31 gene acts near the end of the vulval signaling pathway. We show that lin-31 acts downstream of the ras homolog let-60 and that lin-31 encodes a member of the HNF-3/fork head family of DNA-binding transcription factors. lin-31 regulates how vulval precursor cells choose their fate; in lin-31 mutants, these cells do not properly choose which fate to express and therefore adopt any one of the three possible vulval cell fates in a deregulated fashion. This interesting mutant phenotype suggests mechanisms for how vulval cell fates become determined.
In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.
p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.
Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.
The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.
We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBF-mediated transcription. In the present study, we have examined the mechanism by which Rb represses UBF-dependent rDNA transcription and determined if other Rb-like proteins have similar effects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb.
Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit
and prevent its association with the 40S ribosomal subunit. InSaccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional
mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from
a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing
a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis
indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective
pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased
20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced
formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal
subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin
epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.
Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.
Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene.
We have recently shown that the mammalian nucleolar protein Bop1 is involved in synthesis of the 28S and 5.8S ribosomal RNAs
(rRNAs) and large ribosome subunits in mouse cells. Here we have investigated the functions of the Saccharomyces cerevisiae homolog of Bop1, Erb1p, encoded by the previously uncharacterized open reading frame YMR049C. Gene disruption showed that
ERB1 is essential for viability. Depletion of Erb1p resulted in a loss of 25S and 5.8S rRNAs synthesis, while causing only a moderate
reduction and not a complete block in 18S rRNA formation. Processing analysis showed that Erb1p is required for synthesis
of 7S pre-rRNA and mature 25S rRNA from 27SB pre-rRNA. In Erb1p‐depleted cells these products of 27SB processing are largely
absent and 27SB pre-rRNA is under-accumulated, apparently due to degradation. In addition, depletion of Erb1p caused delayed
processing of the 35S pre-rRNA. These findings demonstrate that Erb1p, like its mammalian counterpart Bop1, is required for
formation of rRNA components of the large ribosome particles. The similarities in processing defects caused by functional
disruption of Erb1p and Bop1 suggest that late steps in maturation of the large ribosome subunit rRNAs employ mechanisms that
are evolutionarily conserved throughout eukaryotes.
The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans. Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23. We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels.
Ribosome biogenesis is a conserved process in eukaryotes that requires a large number of small nucleolar RNAs and trans-acting
proteins. The Saccharomyces cerevisiae MRD1 (multiple RNA-bindingdomain) gene encodes a novel protein that contains five consensus RNA-binding domains. Mrd1p is essential for viability. Mrd1p
partially co-localizes with the nucleolar protein Nop1p. Depletion of Mrd1p leads to a selective reduction of 18 S rRNA and
40 S ribosomal subunits. Mrd1p associates with the 35 S precursor rRNA (pre-rRNA) and U3 small nucleolar RNAs and is necessary
for the initial processing at the A0–A2 cleavage sites in pre-rRNA. The presence of five RNA-binding domains in Mrd1p suggests that Mrd1p may function to correctly
fold pre-rRNA, a requisite for proper cleavage. Sequence comparisons suggest that Mrd1p homologues exist in all eukaryotes.
60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.
A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.
We previously identified Caenorhabditis elegans mutants in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8(n111) II and lin-9(n112) III. In this paper, we identify 18 new mutations, which are alleles of eight genes, that interact with either lin-8(n111) or lin-9(n112) to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The "silent Muv" mutations fall into two classes; hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. Our results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families.
Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The
Saccharomyces cerevisiae
gene that encodes the 245-amino-acid eIF6 (calculated
M
r
25,550), designated
TIF6
, has been cloned and expressed in
Escherichia coli
. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes.
TIF6
is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.
The retinoblastoma gene product has been implicated in the regulation of multiple cellular and developmental processes, including a well-defined role in the control of cell cycle progression. The Caenorhabditis elegans retinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle entry and, as shown by studies of synthetic multivulval genes, plays an important role in the determination of vulval cell fates. We demonstrate an additional and unexpected function for lin-35 in organ morphogenesis. Using a genetic approach to isolate lin-35 synthetic-lethal mutations, we have identified redundant roles for lin-35 and ubc-18 , a gene that encodes an E2 ubiquitin-conjugating enzyme closely related to human UBCH7. lin-35 and ubc-18 cooperate to control one or more steps during pharyngeal morphogenesis. Based on genetic and phenotypic analyses, this role for lin-35 in pharyngeal morphogenesis appears to be distinct from its cell cycle-related functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development.
We previously identified Caenorhabditis elegans mutants in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8(n111) II and lin-9(n112) III. In this paper, we identify 18 new mutations, which are alleles of eight genes, that interact with either lin-8(n111) or lin-9(n112) to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The "silent Muv" mutations fall into two classes; hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. Our results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families.
Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.
We previously identified Caenorhabditis elegans mutants in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8(n111) II and lin-9(n112) III. In this paper, we identify 18 new mutations, which are alleles of eight genes, that interact with either lin-8(n111) or lin-9(n112) to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The "silent Muv" mutations fall into two classes; hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. Our results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families.
Ninety-five mutants of the nematode Caenorhabditis elegans altered in the cell lineages of the vulva have been isolated on the basis of their displaying one of two phenotypes, Vulvaless or Multivulva. In Vulvaless mutants, which define 12 genes, no vulva is present. In Multivulva mutants, which define ten genes, one or more supernumerary vulva-like protrusions are located along the ventral side of the animal. A single recessive mutation is responsible for the phenotypes of most, but not all, of these strains. Fifteen of these 22 genes are represented by multiple alleles. We have shown by a variety of genetic criteria that mutations that result in a Vulvaless or Multivulva phenotype in six of the 22 genes most likely eliminate gene function. In addition, Vulvaless or Multivulva mutations in seven of the other genes most likely result in a partial reduction of gene function; the absence of the activity of any of these genes probably results in lethality or sterility. Our results suggest that we may have identified most, or all, genes of these two classes.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.
A ncl-1 mutation results in enlarged nucleoli, which can be detected in nearly all cells of living animals by Nomarski microscopy. Spontaneous mitotic loss of a ncl-1(+)-containing free duplication in an otherwise homozygous ncl-1 mutant animal results in mosaicism for ncl-1 expression, and the patterns of mosaicism lead us to conclude that ncl-1 acts cell autonomously. The probability of mitotic loss of the duplication sDp3 is approximately constant over many cell divisions. About 60% of the losses of sDp3 at the first embryonic cell division involve nondisjunction. Frequencies of mitotic loss of different ncl-1(+)-bearing free duplications varied over a 200-fold range. The frequencies of mitotic loss were enhanced by a chromosomal him-10 mutation. We have used ncl-1 as a cell autonomous marker in the mosaic analysis of dpy-1 and lin-37. The focus of action of dpy-1 is in hypodermis. A mutation in lin-37 combined with a mutation in another gene results in a synthetic multivulva phenotype. We show that lin-37 acts cell nonautonomously and propose that it plays a role, along with the previously studied gene lin-15, in the generation of an intercellular signal by hyp7 that represses vulval development.
Germ cells complete multiple events to form functional oocytes and sperm. In the Caenorhabditis elegans hermaphrodite, germ cells develop in proximity to the somatic gonad sheath and spermathecal cells. We present evidence from cellular laser ablation studies indicating that cells of the somatic sheath and spermathecal lineages play critical roles in four events of hermaphrodite germline development. (1) Cells of the sheath and spermathecal lineage support germline proliferation; ablation of sheath/spermathecal precursor cells reduces mitotic proliferation. (2) These cells also play a role in the exit of germ cells from the pachytene stage of meiotic prophase and/or gamete differentiation; ablation can result in undifferentiated germ cells arrested in pachytene. (3) Proximal sheath and distal spermatheca cells are required for ovulation of the oocyte. During wild-type ovulation, the mature oocyte is expelled from the gonad arm by contraction of the proximal myoepithelial sheath and dilation of the distal spermatheca. Ablation of these cells traps mature oocytes in the gonad arm where they endomitotically replicate their DNA (the Emo phenotype). (4) Cells of the sheath and spermathecal lineage also appear to promote the male germ cell fate since ablation of one sheath/spermathecal precursor cell can feminize the hermaphrodite germ line. These somatic ablation-induced germline phenotypes demonstrate that the somatic gonad is required for multiple events in C. elegans germline development. Further, these results suggest that soma to germline cell-cell interactions in C. elegans are physiological in character (i.e., contraction during ovulation) as well as regulatory.
This chapter presents the use of minutes in mitotic recombination analysis and in developmental genetic studies and minutes as suppressors are discussed. One of the most important findings described is the isolation of viable homozygous minutes, both fertile and sterile ones. These alleles show a synergistic effect as homozygotes—that is, their phenotype is more severe than in heterozygotes. Northern analysis of all possible combinations of alleles of the same Minute locus is consistent with this observation that less mRNA correlates with a more severe mutant phenotype and vice versa. Recessive alleles will provide a means not only to understand the Minute syndrome but also to study how the Minute genes are regulated. Another important finding is that several ribosomal proteins appear to have other functions apart from being part of the translational machinery—for example, apurinic/apyrimidinic endonuclease activities and tumor-suppressor functions. These results suggest that ribosomal proteins are involved in regulatory processes that may be important in normal development.
The Ras signaling pathway for vulval induction in Caenorhabditis elegans is antagonized by the activity of the synthetic multivulva (synMuv) genes, which define two functionally redundant pathways. We have characterized two genes in one of these pathways. lin-35 encodes a protein similar to the tumor suppressor Rb and the closely related proteins p107 and p130. lin-53 encodes a protein similar to RbAp48, a mammalian protein that binds Rb. In mammals, Rb and related proteins act as regulators of E2F transcription factors, and RbAp48 may act with such proteins as a transcriptional corepressor. We propose that LIN-35 and LIN-53 antagonize the Ras signaling pathway in C. elegans by repressing transcription in the vulval precursor cells of genes required for the expression of vulval cell fates.
Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated Mr 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.
Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. We cloned fog-2 by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3'untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. We propose that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species.
Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.
RNA polymerase I (PolI) transcription is activated by the HMG box architectural factor UBF, which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. Here we show that the acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation "flip-flop." Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb.
We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.
In wild-type Caenorhabditis elegans, the hermaphrodite gonad is a symmetrical structure, whereas the male gonad is asymmetric. Two cellular processes are critical for the generation of these sexually dimorphic gonadal shapes during early larval development. First, regulatory "leader" cells that control tube extension and gonadal shape are generated. Second, the somatic gonadal precursor cells migrate and become rearranged to establish the adult pattern. In this paper, we introduce sys-1, a gene required for early organization of the hermaphrodite, but not the male, gonad. The sys-1(q544) allele behaves genetically as a strong loss-of-function mutant and putative null. All hermaphrodites that are homozygous for sys-1(q544) possess a grossly malformed gonad and are sterile; in contrast, sys-1(q544) males exhibit much later and only partially penetrant gonadal defects. The sys-1(q544) hermaphrodites exhibit two striking early gonadal defects. First, the cell lineages of Z1 and Z4, the somatic gonadal progenitor cells, produce extra cells during L2, but the regulatory cells that control gonadal shape are not generated. Second, somatic gonadal precursor cells do not cluster centrally during late L2, and the somatic gonadal primordium typical of hermaphrodites is not established. In contrast, the early male gonadal lineage is asymmetric as normal, the somatic gonadal primordium typical of males is established correctly, and the male adult gonadal structures can be normal. We conclude that the primary role of sys-1 is to establish the shape and polarity of the hermaphrodite gonad.
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.
A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.
The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.
We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals.
Two new genetic balancers for chromosome II of Caenorhabditis elegans were isolated and characterized. mIn1 was shown to be an inversion of a large central portion of the chromosome, extending from lin-31 to rol-1, that includes most of the genes on the chromosome. It balances a region to the left of the gene cluster that was previously not covered by any of the available balancers. mIn1 recombines efficiently with the normal chromosome II in regions outside the rearrangement at both ends, and appears to enhance recombination frequency adjacent to the inversion breakpoints. Eight variant strains of mIn1 were isolated, including forms that carry recessive morphological or lethal markers, an unmarked form, and one that carries an integrated transgene that confers a semi-dominant green fluorescent protein (GFP) phenotype. This set of variants makes mIn1 useful for a wide variety of applications. The second balancer, mT1, was shown to be a II;III translocation that suppresses recombination on the right arms of chromosomes II and III. It balances chromosome II from the region between bli-2 and dpy-10 to the right end of the chromosome, and chromosome III from the region between daf-2 and unc-93 to the right end. These rearrangements provide the means to stabilize efficiently most of the genes on chromosome II, and may be useful for studies of chromosome pairing and recombination.
We have investigated the role of trigger RNA amplification during RNA interference (RNAi) in Caenorhabditis elegans. Analysis of small interfering RNAs (siRNAs) produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA. Instead, a population of siRNAs (termed secondary siRNAs) appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism. The distribution of secondary siRNAs exhibited a distinct polarity (5'-->3' on the antisense strand), suggesting a cyclic amplification process in which RdRP is primed by existing siRNAs. This amplification mechanism substantially augments the potency of RNAi-based surveillance, while ensuring that the RNAi machinery will focus on expressed mRNAs.
Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.
The regulation of ribosome synthesis is likely to play an important role in the regulation of cell growth. Previously, we have shown that the ncl-1 gene in Caenorhabditis elegans functions as an inhibitor of cell growth and ribosome synthesis. We now indicate that the Drosophila melanogaster tumor suppressor brain tumor (brat) is an inhibitor of cell growth and is a functional homolog of the C. elegans gene ncl-1. The brat gene is able to rescue the large nucleolus phenotype of ncl-1 mutants. We also show that brat mutant cells are larger, have larger nucleoli, and have more ribosomal RNA than wild-type cells. Furthermore, brat overexpressing cells contain less ribosomal RNA than control cells. These results suggest that the tumorous phenotype of brat mutants may be due to excess cell growth and ribosome synthesis.
The single C. elegans member of the retinoblastoma gene family, lin-35 Rb, was originally identified as a synthetic Multivulva (synMuv) gene [1]. These genes form two redundant classes, A and B, that repress ectopic vulval cell fate induction. Recently, we demonstrated that lin-35 Rb also acts as a negative regulator of G(1) progression and likely is the major target of cyd-1 Cyclin D and cdk-4 CDK4/6. Here, we describe G(1) control functions for several other class B synMuv genes. We found that efl-1 E2F negatively regulates cell cycle entry, while dpl-1 DP appeared to act both as a positive and negative regulator. In addition, we identified a negative G(1) regulatory function for lin-9 ALY, as well as lin-15B and lin-36, which encode novel proteins. Inactivation of lin-35 Rb, efl-1, or lin-36 allowed S phase entry in the absence of cyd-1/cdk-4 and increased ectopic cell division when combined with cki-1 Cip/Kip RNAi. These data are consistent with lin-35 Rb, efl-1, and lin-36 acting in a common pathway or complex that negatively regulates G(1) progression. In contrast, lin-15B appeared to act in parallel to lin-35. Our results demonstrate the potential for genetic identification of novel G(1) regulators in C. elegans.