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Assembled U4/U6 snRNP accumulates in Cajal bodies after hPrp22 and hNtr1 knockdown. (A) Cells treated with siRNA against hPrp22 (22-3) were transfected with SART3-YFP and hPrp31-CFP, and FRET was measured by acceptor photobleaching in the nucleoplasm and CBs. (B) Quantification of SART3-YFP/hPrp31-CFP FRET measurements in cells treated with control (NC), hPrp22-3, or hNtr1-27 siRNAs. Average values of 10 measurements with SE bars are shown.
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The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluoresce...
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... was measured as described previously in the nucleoplasm and CBs by acceptor photo- bleaching ( Stanek and Neugebauer, 2004). No significant changes were observed in CBs after treatment with anti- hPrp22 or anti-hNtr1 siRNAs (Figure 7), showing that U4/U6 components accumulating in CBs are assembled into the U4/U6 snRNP. These data indicate that inhibition of spliceosome recycling leads to specific accumulation of the U4/U6 snRNPs in CBs. ...
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The spliceosome is a dynamic macromolecular machine that assembles on pre-messenger RNA substrates and catalyses the excision of non-coding intervening sequences (introns). Four of the five major components of the spliceosome, U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs), contain seven Sm proteins (SmB/B', SmD1, SmD2, SmD3, SmE, SmF...
Citations
... Nevertheless, separate binding of SANS to the two PRPFs could also occur during the formation of the tri-snRNP complex in the Cajal bodies when the U4/U6 snRNP containing PRPF31 and U5 snRNP harboring PRPF6 maturate separately from each other [41]. Alternatively, in the tri-snRNP complex, SANS may preferentially bind to PRPF31, as indicated by our FRET data, and interacts with PRPF6 during the recycling process of the tri-snRNP complex components after activation of the spliceosome when sufficient free U5 snRNP/PRPF6 is present [2,42]. Although the latter alternative is supported by previous work showing that SANS deficiency does not affect the maturation of the tri-snRNP complex but does affect the recycling of U5-snRNP after activation of the spliceosome [3], in the tri-snRNP complex, the SANS binding site of PRPF31 might already be occupied by SNU13 and U4-snRNA, which could interfere with the binding of SANS [28]. ...
Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf–blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.
... Nevertheless, separate binding of SANS to the two PRPFs could also occur during the formation of the tri-snRNP complex in the Cajal bodies when the U4/U6 snRNP containing PRPF31 and U5 snRNP harboring PRPF6 maturate separately from each other [47]. Alternatively, in the tri-snRNP complex, SANS may preferentially bind to PRPF31 as indicated by our FRET data and interacts with PRPF6 during the recycling process of the tri-snRNP complex components after activation of the spliceosome when the sufficient free U5 snRNP/PRPF6 is present [2,48]. Although the latter alternative is supported by previous work showing that SANS deficiency does not affect the maturation of the tri-snRNP complex but the recycling of U5-snRNP after activation of the spliceosome (Yildirim et al., 2021), in the tri-snRNP complex the SANS binding site of PRPF31 might already be occupied by SNU13 and U4-snRNA which could interfere with the binding of SANS [34]. ...
Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intra-nuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH) the most common form of hereditary deaf-blindness.Here, we aimed to gain further insight into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by binding of PRPF6. Furthermore, we found that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs implying a significance for the USH1G pathophysiology.
... Most of the available data point to a function of CB in sn/snoRNP metabolism, namely the enhancement of snRNP assembly [49,[92][93][94][95] (Figure 2). The strongest evidence came from experiments in developing zebrafish embryos, where the lethal phenotype induced by coilin depletion was rescued by injection of mature snRNPs [39]. ...
... My laboratory and others have shown that inhibition of the final steps of snRNP biogenesis results in sequestration of various snRNP assembly intermediates in CB [49,74,93,95,[99][100][101][102]. Similarly, inhibition of snRNP recycling after splicing increases the accumulation of specific snRNPs in CB [93,103], which is consistent with the view that CB are involved in snRNP reassembly after splicing and quality control of the snRNP (re)assembly process. ...
... My laboratory and others have shown that inhibition of the final steps of snRNP biogenesis results in sequestration of various snRNP assembly intermediates in CB [49,74,93,95,[99][100][101][102]. Similarly, inhibition of snRNP recycling after splicing increases the accumulation of specific snRNPs in CB [93,103], which is consistent with the view that CB are involved in snRNP reassembly after splicing and quality control of the snRNP (re)assembly process. The molecular mechanism that discriminates between assembly intermediates and fully mature particles is currently unclear. ...
The nucleus of higher eukaryotes contains a number of structures that concentrate specific biomolecules and play distinct roles in nuclear metabolism. In recent years, the molecular mechanisms controlling their formation have been intensively studied. In this brief review, I focus on coilin and Cajal bodies. Coilin is a key scaffolding protein of Cajal bodies that is evolutionarily conserved in metazoans. Cajal bodies are thought to be one of the archetypal nuclear structures involved in the metabolism of several short non-coding nuclear RNAs. Yet surprisingly little is known about the structure and function of coilin, and a comprehensive model to explain the origin of Cajal bodies is also lacking. Here, I summarize recent results on Cajal bodies and coilin and discuss them in the context of the last three decades of research in this field.
... Despite the high concentration of splicing factors, Cajal bodies and speckles are not the sites where splicing predominantly takes place. Cajal bodies constitute sites of small nuclear (sn) RNP biogenesis, where the assembly of the U4/U6 di-snRNP and the U4/U6-U5 tri-snRNP, the final maturation steps of the U2 snRNP as well as 2′-O-methylation and pseudo-uridylation of snRNAs have been shown to occur [9][10][11] . Furthermore, the tri-snRNP is re-assembled in Cajal bodies after its major remodeling during each round of splicing 9 . ...
... Cajal bodies constitute sites of small nuclear (sn) RNP biogenesis, where the assembly of the U4/U6 di-snRNP and the U4/U6-U5 tri-snRNP, the final maturation steps of the U2 snRNP as well as 2′-O-methylation and pseudo-uridylation of snRNAs have been shown to occur [9][10][11] . Furthermore, the tri-snRNP is re-assembled in Cajal bodies after its major remodeling during each round of splicing 9 . Speckles are largely devoid of nascent RNAs 12,13 and are thought to represent storage sites for splicing factors. ...
... rCID was also differentially effective in targeting splicing factors in different sub-nuclear regions. Splicing factors continuously cycle between Cajal bodies, speckles, the nucleoplasm, and perichromatin 9,22 . Cajal bodies and speckles themselves represent highly dynamic structures, undergoing movements within the inter-chromatin space and associating with actively transcribed genes and chromatin regions 21,22,25,60,61 . ...
Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments. Through the use of a reversible chemical dimerizer, the splicing factor PRPF38A is re-localized to the nuclear lamina, paving the way for a systematic analysis of spatio-temporal splicing regulation.
... As another example in humans, the 20S U5 snRNP associates with the pre-mRNA-processing factor (PRPF) 19 complex and additional factors during splicing, ultimately being released as a 35S particle (17), from which the U5 snRNP has to be regenerated. Like de novo snRNP biogenesis, recycling of snRNPs after splicing requires specialized molecular machinery and is also thought to take place in Cajal bodies (14,15,18,19). ...
Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling.
... By exerting quality control over the stability and function of a variety of cellular RNAs, including tRNAs, rRNAs, and the RNA constituents of the spliceosome, the H/ACA complex possesses broad functionality in promoting fidelitous gene expression. For instance, TCAB1 recruits telomerase to CBs, which are sites of assembly for spliceosomal snRNPs [128,129]. Furthermore, CBs appear to nucleate chromosomal clusters that contain genomic loci rich in histone genes, thereby influencing their expression [130]. Coupled with the presence of histone proteins in the telomerase holoenzyme, these findings emphasize the critical functional diversity of all of the telomerase accessory components. ...
Simple Summary
Cells undergoing malignant transformation must circumvent replicative senescence and eventual cell death associated with progressive telomere shortening that occurs through successive cell division. To do so, malignant cells reactivate telomerase to extend their telomeres and achieve cellular immortality, which is a “Hallmark of Cancer”. Here we review the telomere-dependent and -independent functions of telomerase in cancer, as well as its potential as a biomarker and therapeutic target to diagnose and treat cancer patients.
Abstract
During the process of malignant transformation, cells undergo a series of genetic, epigenetic, and phenotypic alterations, including the acquisition and propagation of genomic aberrations that impart survival and proliferative advantages. These changes are mediated in part by the induction of replicative immortality that is accompanied by active telomere elongation. Indeed, telomeres undergo dynamic changes to their lengths and higher-order structures throughout tumor formation and progression, processes overseen in most cancers by telomerase. Telomerase is a multimeric enzyme whose function is exquisitely regulated through diverse transcriptional, post-transcriptional, and post-translational mechanisms to facilitate telomere extension. In turn, telomerase function depends not only on its core components, but also on a suite of binding partners, transcription factors, and intra- and extracellular signaling effectors. Additionally, telomerase exhibits telomere-independent regulation of cancer cell growth by participating directly in cellular metabolism, signal transduction, and the regulation of gene expression in ways that are critical for tumorigenesis. In this review, we summarize the complex mechanisms underlying telomere maintenance, with a particular focus on both the telomeric and extratelomeric functions of telomerase. We also explore the clinical utility of telomeres and telomerase in the diagnosis, prognosis, and development of targeted therapies for primary, metastatic, and recurrent cancers.
... Enamel formation gene. ought to interact with tuftelin and can play a role in spliceosome disassembly in Cajal bodies [32]. ...
Objectives:
The present review aims to give an overview of the literature focusing on novel genetic aspects of dental erosion and dental caries. Once the tooth erupts into the oral cavity, the regenerative capability of enamel is fundamentally limited due to the loss of dental epithelium during eruption. The susceptibility or resistance to dental erosion and caries is presumably a result of environmental, phenotypic, and/or genetic influence. Even though it is evident that individuals frequently exposing their teeth to acid and sugar are at high risk of developing dental erosion and caries, the findings exclusively based on these factors are elusive. Data resources and study selection. The present review was based on data collected from the National Library of Medicine database with different combinations of the following terms: "tooth," "dental," "dentin," "enamel," "erosion," "erosive wear," "caries," "decay," "gene," and "genetic." A total of forty-six studies met the inclusion criteria. Data were extracted by one reviewer and verified by another.
Conclusion:
The high prevalence of erosion and caries among certain groups, and observations that not all individuals appearing to be at risk develop these lesions, has sparked research on identifying genetic effects to these conditions. A connection of genome-wide and candidate gene studies has increased considerably in the literature. This review reveals largely varying success among studies, demonstrating the difficulties of developing the study with adequate sample sizes and durable phenotype definitions that permit enough statistical power to identify genetic contributors.
... For example, inhibition of U5 snRNP maturation by PRPF8 or R2TP downregulation results in the accumulation of U5, U4, and U6 snRNAs in Cajal bodies 7,12 . In contrast, inhibition of post-spliceosomal complex recycling caused specific accumulation of U4 and U6 snRNAs but not U5 snRNA 45 . To test a function of TSSC4 in U5 snRNP biogenesis and recycling, we reduced TSSC4 expression by RNA interference and assayed localization of snRNAs in Cajal bodies (Fig. 5). ...
U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.
... Strikingly, in contrast to U4/U6 components, U5 snRNP-specific proteins and snRNA did not accummulate in Cajal bodies in SANS deficient cells (Figure 4). This phenomenon was also observed in a previous study after inhibition of spliceosome disassembly, which was associated with insufficient recycling and delivery of U5 snRNPs back to Cajal bodies (81). Therefore, it is likely that SANS also plays a role in the recycling pathway of splicing molecules, which is also consistent with its interaction with core spliceosome components. ...
Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
... For decades, the CB has been presumed to be the location of assembly and/or modification of the splicing snRNPs, since they are enriched in proteins and RNAs (U1 to U6) involved in splicing (Carmo-Fonseca et al., 1992;Matera and Ward, 1993;Stanĕk et al., 2003;Schaffert et al., 2004;Staněk et al., 2008). ...
Ancestral environmental conditions can instruct offspring development, although the mechanism(s) underlying such transgenerational epigenetic inheritance is unclear. In murine models focused on paternal dietary effects, we and others have identified tRNA fragments (tRFs) in mature sperm as potential carriers of epigenetic information. In our search for molecular targets of specific tRFs, we observed that altering the level of 5’-tRF Glycine-GCC (tRF-GG) in mouse embryonic stem cells (mESCs) and preimplantation embryos modulates the expression of the endogenous retrovirus MERV-L and genes regulated by MERV-L. Intriguingly, transient derepression of MERV-L is associated with totipotency of two-cell stage embryos and a subset of two-cell-like mESCs. Here, I reveal the mechanistic basis for tRF-GG regulation of MERV-L. I show that tRF-GG supports the production of numerous small nuclear RNAs associated with the Cajal body, in mouse and human embryonic stem cells. In particular, tRF-GG modulates the levels of U7 snRNA to ensure an adequate supply of histone proteins. This in turn safeguards heterochromatin-mediated transcriptional repression of MERV-L elements. Importantly, tRF-GG effects on histone mRNA levels, activity of a histone 3’UTR reporter, and expression of MERV-L associated transcripts can all be suppressed by appropriate manipulation of U7 RNA levels. I also show that hnRNPF and H bind directly to tRF-GG, and display a stark overlap of in vivo functions to tRF-GG. Together, this data uncovers a conserved mechanism for a 5’ tRNA fragment in the fine-tuning of a regulatory cascade to modulate global chromatin organization during pre-implantation development.
