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Single gene analysis of copy number effect on RNA interference (RNAi). (A) Schematic of single-copy and multi-copy MAL32 system. (B) Northern analysis of MAL32 RNA from single-copy and multi-copy systems. All visible species are included in antisense quantification. (C) MAL32 short interfering RNA (siRNA) abundance from cells in B. (D) RNase A sensitivity of MAL32 in wild-type cells expressing multi-copy and single-copy MAL32. Cells were lysed on ice, treated with RNase A as indicated and analyzed by northern blot. 25S and L-A (a double stranded RNA) are shown as controls for loading and RNase specificity. n = 3 biological replicates, error bars ±1se, *p<0.05, ***p<0.01 by Student's t test, y axes in arbitrary units. DOI: 10.7554/eLife.01581.018
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... following figure supplements are available for figure 3: the single-strand specific nuclease RNase A, and observed significantly more RNase-resistant material in cells expressing MAL32 from the multi-copy system than the single-copy system ( Figure 5D). This experiment shows that a multi-copy locus produces more dsRNA than an equivalently expressed single-copy locus in wild-type cells without the RNAi system, explaining the increased siRNA formation in RNAi+ ...
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... then directly tested the effect of copy number at the MAL32 locus. We constructed strains in which MAL32 sense and antisense RNAs were expressed at similar levels from multi-copy or single-copy loci by over-expressing single-copy sense and antisense ( Figure 5A). In this system, both sense and antisense RNAs were produced at higher levels from the single-copy system (Figure 5B compare lanes 1 and 3) but more siRNAs were produced from the multi-copy system ( Figure 5C compare lanes 2 and 4). The over-expression of both RNAs from the single-copy MAL32 locus led to the production of easily detectable siRNA, as would be expected; however, this result directly demonstrates that gene copy number influences the formation of siRNA above and beyond the effect of total RNA abundance. The increased siRNA production in these cells is most likely due to enhanced dsRNA formation in the multi-copy system. To confirm this, we quantified MAL32 RNA in wild-type cells that is resistant to cells from C. (E) Schematic of GAL4 locus. (F) GAL4 mRNA and antisense ncRNA in wild-type and RNAi+ strains; cells grown in YP galactose (extended image and quantification shown in Figure 3-figure supplement 2B). (G) mRNA and antisense ncRNA from GAL4 locus cloned onto a high-copy plasmid in wild-type and RNAi + strains. Lanes 5,6 show empty vector, signal is from genomic GAL4; note that cells used here are diploids to mitigate defects in galactose response (see 'Materials and methods'). Lanes 3,4 show a previously described GAL4 antisense mutant (Geisler et al., 2012); this removes detectable antisense RNA for genomic GAL4, but the mutant sequence still expresses an antisense ncRNA when cloned on the high-copy plasmid (see Figure 3-figure supplement 4). (H) siRNA analysis of cells in G. For quantification, n = 4 biological replicates, error bars represent ± 1se, *p<0.05, ***p<0.01 by Student's t test, y axes in arbitrary units. DOI: ...
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... siRNAs produced from the high-copy GAL10 locus are clearly sufficient to degrade the GAL10 ncRNAs in the RNAi+ background ( Figure 8B compare lanes 5,7 with lanes 6,8); however, a classical RNAi response should be able to degrade RNA expressed from a separate locus. To test this we intro- duced the high-copy GAL cluster plasmids into a strain in which the single-copy genomic GAL10 ORF is expressed at high levels from a Cu 2+ -dependent promoter, allowing expression of the GAL10 mRNA from the single-copy locus while the GAL clusters present on the high-copy plasmids remain fully repressed. As observed for the GAL10 ncRNAs, the GAL10 mRNA was expressed at higher levels in the RNAi+ strain than in the wild-type ( Figure 8D compare lanes 1,2) but, nonetheless, both wild-type and RBSΔ high-copy GAL cluster plasmids caused highly significant >50% knockdowns of the GAL10 mRNA compared with the empty vector control ( Figure 8D lanes 2,4,6). This was not an indirect effect of the high-copy GAL clusters alone as, in the wild-type background, GAL10 mRNA levels were slightly increased by the presence of the GAL plasmids ( Figure 8D lanes 1,3,5). These data demonstrate that pervasive transcription of a high-copy locus is sufficient to instigate an effective RNAi response that can mediate the degradation of a target mRNA in ...
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... following figure supplements are available for figure 4: Reb1 binding site mutant (RBSΔ), leaving almost no detectable RNAs from this locus ( Figure 8B lane 3). For reasons that remain unclear, the GAL cluster is slightly de-repressed in the RNAi+ strain ( Figure 8B compare lanes 1,2); nonetheless, the RBSΔ RNAi+ strain ( Figure 8B lane 4) only produces very low level heterogeneous transcripts from GAL10, suggesting that it forms a good model of per- vasive transcription. Cloning either wild-type or RBSΔ GAL clusters onto high-copy plasmids substan- tially increased the levels of detectable ncRNA as expected ( Figure 8B lanes 5,7), and these ncRNAs were processed into easily detectable siRNAs ( Figure 8C lanes 6,8). Therefore, ncRNAs produced at the level of pervasive transcription are sufficient to mediate extensive siRNA production when the copy number of the transcribing locus is ...
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... The upper phase was re-extracted first with 500 l phenol:chloroform pH 7 (1:1) and then with 500 l chloroform:isoamyl alcohol (24:1) before precipitation with 1 ml ethanol. Pellet was washed with 70% ethanol, re-suspended in 6 l water and quantified using Quant-IT RiboGreen (ThermoFisher, R11490). 1 g RNA was resolved per lane on 1.2% glyoxal agarose gels, blotted and probed with a random primed probe against CUP1 ORF (Supplementary Table S2) as described (61). ...
Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.
... For transcript levels, little work has been down. The only study finds that endogenous RNAi is driven by copy number because higher level of transcripts makes dsRNA generation easier [35]. Thus, in this work we explored the influence of transcript level to exogenous RNAi efficiency by checking expression knockdown. ...
Efficiency is the base for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly with insect species, tissues and genes. Previous efforts have revealed the mechanisms for variations among insect species and tissues. Here, we investigated the reason for variable efficiency among the genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression level and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.
... The selectivity of such regulation would be controlled by specific RNA-binding proteins and/or non-coding RNAs. Curiously, experiments in yeasts have shown that mRNA of genes with an increased copy number expressed from endogenous loci can be selectively targeted and degraded by RNA interference 57 . A similar mechanism might be responsible for a decrease in mRNA levels of multi-copy transgenes in CHO cells. ...
The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multi-copy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5’ and 3’ gene regulatory elements and landing pad features on qP. We evaluated the limitations of multi-copy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multi-copy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multi-copy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.
... There are a few limitations to be aware of particularly that many column-based methods do not isolate RNA of less thañ 200 nt. For budding yeast, kits are less widely used but GTC/phenol and hot phenol methods work well, and the mir-VANA protocol can be readily adapted using glass bead lysis (see, e.g., [18] for all three methods). RNA can be further manipulated for example by poly(A)+ purification or RNase H cleavage. ...
Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide–urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.
... However, upon heterologous expression in S. cerevisiae of RNAi factors from the close RNAicapable relative species Naumovozyma castellii ( Drinnenberg et al. 2009;Drinnenberg et al. 2011), we observed a massive production of siRNAs from asXUTs, indicating that they can form dsRNA structures with their paired-sense mRNAs in vivo (Wery et al. 2016). Consistent with this observation, N. castellii Dicer was detected in the cytoplasm when expressed in S. cerevisiae (Cruz and Houseley 2014). ...
... Since asXUTs (ie the aslncRNAs that are degraded in the cytoplasm) constitute the preferred targets of Dcr1 for small RNAs production, we anticipated that Dcr1 localizes in the cytoplasm. Further supporting this hypothesis, Dcr1 was previously detected as cytoplasmic foci when artificially expressed as a fusion with the GFP in S. cerevisiae (Cruz and Houseley 2014). ...
... The idea that Dcr1 and Xrn1 functionally interact is reinforced by the observation that Dcr1 localizes in the cytoplasm (Figure 4), which is consistent with previous observations made upon expression of a Dcr1-GFP fusion in S. cerevisiae (Cruz and Houseley 2014). Moreover, among the different classes of aslncRNAs, the asXUTs constitute the preferred target for small RNAs production ( Figure 3A). ...
Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.
... Cells were diluted to 0.06x10 7 cells/ml in 12.5 ml YPD and grown for 1.5 hours before addition of 12.5 ml YPD with or without 2 mM nicotinamide (Sigma N3376) and grown for 6 hours before harvesting 2x10 7 cells by centrifugation and freezing on N2. RNA was extracted by the GTC phenol miniprep method, and 1.5 µl of 6 µl eluate run on 1% BPTE gels, imaged and blotted as described (Cruz and Houseley, 2014). Gels were probed for BNA4 and BNA6 then re-probed for BNA1, or for BNA5 and BNA7, in UltraHyb (Life Technologies AM8670) using RNA probes listed in Table S5 as described (Cruz and Houseley, 2014). ...
... RNA was extracted by the GTC phenol miniprep method, and 1.5 µl of 6 µl eluate run on 1% BPTE gels, imaged and blotted as described (Cruz and Houseley, 2014). Gels were probed for BNA4 and BNA6 then re-probed for BNA1, or for BNA5 and BNA7, in UltraHyb (Life Technologies AM8670) using RNA probes listed in Table S5 as described (Cruz and Houseley, 2014). ...
Transcription of protein coding genes is accompanied by recruitment of COMPASS to promoter-proximal chromatin, which deposits di- and tri-methylation on histone H3 lysine 4 (H3K4) to form H3K4me2 and H3K4me3. Here we determine the importance of COMPASS in maintaining gene expression across lifespan in budding yeast. We find that COMPASS mutations dramatically reduce replicative lifespan and cause widespread gene expression defects. Known repressive functions of H3K4me2 are progressively lost with age, while hundreds of genes become dependent on H3K4me3 for full expression. Induction of these H3K4me3 dependent genes is also impacted in young cells lacking COMPASS components including the H3K4me3-specific factor Spp1. Remarkably, the genome-wide occurrence of H3K4me3 is progressively reduced with age despite widespread transcriptional induction, minimising the normal positive correlation between promoter H3K4me3 and gene expression. Our results provide clear evidence that H3K4me3 is required to attain normal expression levels of many genes across organismal lifespan.
... In contrast to somatic cells, increased pervasive transcription across TEs was reported in ESCs, suggesting that TEs may regulate transcription of long noncoding RNAs (lncRNAs) (Kelley and Rinn, 2012). Intriguingly, however, in yeast it was shown that genome-wide pervasive transcription antisense to transposons leads to an RNAi response as a defense mechanism against TEs (Cruz and Houseley, 2014). Sense/antisense transcription permits the production of double-stranded RNA (dsRNA) triggering RNAi (Fire et al., 1998), which has also been identified as a control mechanism of TEs (Robert et al., 2005). ...
Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a “trap” that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.
... Earlier research has discovered increased pervasive transcription over TEs in ESCs in comparison to somatic cells (Kelley and Rinn, 2012). It was shown in yeast, that low level of genome-wide pervasive transcription, antisense to genic transcription, can initiate RNAi as a defence mechanism against TEs (Cruz and Houseley, 2014). ...
... Earlier studies in yeast have shown that low level of genome-wide pervasive transcription, antisense to genic transcription, can initiate RNAi as a defence mechanism against TEs (Cruz and Houseley, 2014). ...
... The TE sense transcript and the genic sense transcript can produce dsRNAs and can feed into an endosiRNA pathway and controling retrotransposition. This model is supported by findings in the yeast genome (Cruz and Houseley, 2014). The produced endosiRNA are going through an AGO2 dependent mechanism and are potentially involved in TE regulation subsequent to their initial transcriptional activation. ...
DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.
... A list of primers used can be found in Additional file 3: Table S2. For northern blotting, 0.5 μg of glyoxylated total RNA was separated on a 1.2% BPTE gel, then blotted and probed as described in [58]. RNA probes were amplified from mouse genomic DNA using the primers detailed in Additional file 3. ...
Background
Ten-eleven translocation (TET) enzymes oxidise DNA methylation as part of an active demethylation pathway. Despite extensive research into the role of TETs in genome regulation, little is known about their effect on transposable elements (TEs), which make up nearly half of the mouse and human genomes. Epigenetic mechanisms controlling TEs have the potential to affect their mobility and to drive the co-adoption of TEs for the benefit of the host. ResultsWe performed a detailed investigation of the role of TET enzymes in the regulation of TEs in mouse embryonic stem cells (ESCs). We find that TET1 and TET2 bind multiple TE classes that harbour a variety of epigenetic signatures indicative of different functional roles. TETs co-bind with pluripotency factors to enhancer-like TEs that interact with highly expressed genes in ESCs whose expression is partly maintained by TET2-mediated DNA demethylation. TETs and 5-hydroxymethylcytosine (5hmC) are also strongly enriched at the 5′ UTR of full-length, evolutionarily young LINE-1 elements, a pattern that is conserved in human ESCs. TETs drive LINE-1 demethylation, but surprisingly, LINE-1s are kept repressed through additional TET-dependent activities. We find that the SIN3A co-repressive complex binds to LINE-1s, ensuring their repression in a TET1-dependent manner. Conclusions
Our data implicate TET enzymes in the evolutionary dynamics of TEs, both in the context of exaptation processes and of retrotransposition control. The dual role of TET action on LINE-1s may reflect the evolutionary battle between TEs and the host.
... Mechanistically, dosage can affect gene transcription or RNA stability through simple or combined trans-regulatory changes affecting transcription factors and small RNAs (Birchler and Veitia, 2010;Carlsbecker et al., 2010;Cruz and Houseley, 2014;Mallory et al., 2004;Veitia et al., 2013). These effects are often directly proportional to dosage (Huettel et al., 2008;Horiguchi et al., 2009), but compensatory or dosage-inverse changes have been observed as well (Birchler et al., 2001(Birchler et al., , 2007Huettel et al., 2008;Birchler and Veitia, 2010). ...
Altering gene dosage through variation in gene copy number is a powerful approach to addressing questions regarding gene regulation, quantitative trait loci, and heterosis, but one that is not easily applied to sexually transmitted species. Elite poplar (Populus spp) varieties are created through interspecific hybridization, followed by clonal propagation. Altered gene dosage relationships are believed to contribute to hybrid performance. Clonal propagation allows for replication and maintenance of meiotically unstable ploidy or structural variants and provides an alternative approach to investigating gene dosage effects not possible in sexually propagated species. Here, we built a genome-wide structural variation system for dosage-based functional genomics and breeding of poplar. We pollinated Populus deltoides with gamma-irradiated Populus nigra pollen to produce >500 F1 seedlings containing dosage lesions in the form of deletions and insertions of chromosomal segments (indel mutations). Using high-precision dosage analysis, we detected indel mutations in ∼55% of the progeny. These indels varied in length, position, and number per individual, cumulatively tiling >99% of the genome, with an average of 10 indels per gene. Combined with future phenotype and transcriptome data, this population will provide an excellent resource for creating and characterizing dosage-based variation in poplar, including the contribution of dosage to quantitative traits and heterosis.
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