Spt4 promotes Pol I processivity and transcription elongation
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE166983 | GEO | 2021/02/18
REPOSITORIES: GEO
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Project description:Spt4 promotes Pol I processivity and transcription elongation
| PRJNA702517 | ENA
Project description:Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in the absence of Spt5, a fraction of Pol II molecules are dislodged during elongation thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15-20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed and processivity. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning towards maximum elongation speed 15-20 kb from the promoter.
2018-03-06 | GSE106313 | GEO
Project description:Aim and Methods: To understand the precise role of Spt4 in budding yeast, various high throughput sequencing techniques were used. Nucleosome positions were studied using MNase-seq in WT and spt4∆ cells. The position of RNAPII was examined using NET-seq in spt4∆ cells or cells in which Spt4 has been anchored away to the cytoplasm (Spt4 AA). RNAPII was also mapped in cells in which Spt5 has been anchored away to the cytoplasm (Spt5 AA). Steady-state transcript levels were assessed using RNA-seq in samples coupled with NET-seq. The position of the Spt4 on RNAPII was mapped at single nucleotide resolution using TEF-seq. With the same technique, the position of Spt5 on RNAPII was investigated in wild type (WT) and spt4 knock-out (spt4∆) cells. The position and levels of the pre-initiation complex were assessed using ChIP-seq on Sua7(TFIIB) in WT and spt4∆ cells. Results: The interaction between the Spt4/5 complex and RNAPII periodically changes as RNAPII transitions through nucleosomes. The dynamic interaction of Spt5 with RNAPII is dependent on Spt4. In spt4∆ and Spt4 AA cells, RNAPII distribution is altered in a similar way compared to their respective controls. After transcribing into nucleosomes, RNAPII accumulates upstream of nucleosome dyads, especially around the +2 nucleosome. In Spt5 AA cells, the distribution and amount of RNAPII on genes are severely affected. Nucleosome positions are altered in spt4∆ cells. Although the position of the +1 nucleosome remains unchanged, nucleosome spacing from this point is increased, and the level of increase in nucleosome spacing correlates with the level of RNAPII accumulation. Conclusion: This work suggests that Spt4 regulates nucleosome positioning by a mechanism related to transcription and promotes RNAPII movement through nucleosome barriers, especially the barrier set by the +2 nucleosome.
2021-09-27 | GSE159291 | GEO
Project description:The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity. Experiment Overall Design: The expression pattern of Spt5 knockdown embryos was compared to that of the control mopholino oligo injected embryos, both at 21 hours post-fertilization, to show the targets of Spt5. The activation levels of the Spt5-knockdown affected genes were measured by compareing the abundance of transcripts in 21 hours post-fertilization untreated embryos to that in 5 hours post-fertilization untreated embryos.
2008-06-14 | E-GEOD-6127 | biostudies-arrayexpress
Project description:Within the first 15 minutes of infection, herpes simplex virus 1 (HSV-1) immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export of viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi) we observed Increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi Pol II accumulation on specific mutant viral genes was higher than wt virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants, and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.
2024-04-24 | GSE242636 | GEO
Project description:we performed Native Elongating Transcript Sequencing (NET-seq) on an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy throughout the 35S gene and the IGS. Compared to WT, we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Occupancy of rpa12Δ Pol I increased just upstream of the promoter proximal Reb1 binding site and dropped significantly after, implicating this site as a third terminator for Pol I transcription.
2019-12-21 | GSE142457 | GEO
Project description:The Spt4-Spt5 complex, and its human homolog DSIF (DRB sensitivity-inducing factor), is unique in its ability to regulate Pol II processivity. Previous studies have shown that Spt5 has the characteristics of a general transcription-elongation factor. However, mutagenesis of Spt5 showed specific phenotypes during development, which were far less severe than those of Pol II defects or TBP deficient embryos. It seems paradoxical that a mutation which alters a general elongation factor can cause rather specific developmental defects. By using Spt5 knockdown zebrafish embryos and microarrays, here we showed that transcript abundance for only a small subset of genes is altered by loss of Spt5. Further investigation of the down-regulated genes showed that the genes most intensely repressed by the knockdown were strongly activated during early development in untreated embryos. Thus, this study shows that gene activation levels may create different requirements for Pol II processivity. Active transcription requires Spt5 for efficient elongation through its stimulatory activity on Pol II processivity. Keywords: Spt5, microarray analysis, development, transcription, transcription elongation
2007-10-25 | GSE6127 | GEO
Project description:The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline (micronucleus - MIC) and somatic nuclei (macronucleus - MAC), massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. As we were mostly interested in transcription of the germline genome in the MIC during meiosis, we decided to probe the composition of the Spt5-Spt4 complex characteristic to this nucleus. Experiments in which Spt4mB-3xFLAG was used as bait demonstrated its interaction with either Spt5m or Spt5v, which is consistent with the Spt4mB-GFP localization pattern. Interestingly, mass spectrometry revealed that Spt5m-3xFLAG (which is present only in the MIC at the analyzed stage) interacts either with the meiotic Spt4mB or with Spt4vA. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.
2022-04-27 | PXD027748 | Pride
Project description:RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.
| S-EPMC8000598 | biostudies-literature
Project description:To sustain growth, budding yeast actively transcribes its ribosomal gene array (rDNA) in the nucoleolus to produce ribosomes and proteins. However, intense transcription during rDNA replication may provoke collisions between RNA polymerase I (Pol I) and the replisome, may cause replication fork instability, double-strand breaks, local recombinations and rDNA instability. The latter is manifested by rDNA array expansion or reduction and the formation of extrachromosomal rDNA circles, anomalies that accelerate aging in yeast. Transcription also interferes with the resolution, condensation and segregation of the sister chromatid rDNA arrays. As a consequence, rDNA segregation lags behind the rest of the yeast genome and occurs in late anaphase when rDNA transcription is temporarily shut off. How yeast promotes the stability and transmission of its rDNA array while satisfying a constant need for ribosomes remains unclear. Here we show that the downregulation of Pol I by the conserved cell cycle kinase Rio1 spatiotemporally coordinates rDNA transcription, replication and segregation. More specifically, Rio1 activity promotes copy-number stability of the replicating rDNA array by curtailing Pol I activity and by localising the histone deacetylase Sir2, which establishes a heterochromatic state that silences rDNA transcription. At anaphase entry, Rio1 and the Cdc14 phosphatase target Pol I subunit Rpa43 to dissociate Pol I from the 35S rDNA promoter. The rDNA locus then condensates and segregates, thereby concluding the genome transmission process. Rio1 is involved in ribosome maturation in the cytoplasm of budding yeast and human cells. Additional engagements in the cytoplasm or roles in the nucleus are unknown. Our study describes its first nuclear engagement as a Pol I silencing kinase. This activity may prove highly relevant as dysregulated RNA polymerase I activity has been associated with cancer initiation and proliferation.
2017-02-10 | GSE54930 | GEO