Spt4 promotes Pol I processivity and transcription elongation
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ABSTRACT:
PROVIDER: PRJNA702517 | ENA |
REPOSITORIES: ENA
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Project description:The goal of this study was to determine the effect of Spt4 on transcription by Pol I in vivo. Therefore, Native Elongating Transcript Sequencing (NET-seq) was used to evaluate the occupancy of Pol I in wild-type and spt4△ strains. We determined that in spt4△ yeast, there was a processivity defect and Pol I accumulated at the 5' end of the 35S gene. Additionally, we observed that there was an enrichment for G-rich sequences just downstream of a high A content in the spt4△ strain, indicating that perhaps Spt4 plays a role in helping to propel Pol I through these regions of the rDNA. Altogether, this study suggests that Spt4 is an important transcription factor for Pol I by promoting polymerase processivity.
2021-02-18 | GSE166983 | GEO
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: Not available
| S-EPMC8000598 | biostudies-literature
Project description:mRNA cap addition occurs early during RNA pol II transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription, independently of mRNA capping and translation. In cells, sub-lethal suppression of RNMT-RAM reduces RNA pol II occupancy, net mRNA synthesis and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independently of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA-binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription associated factors including RNA pol II subunits, SPT4, SPT6 and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2B K120 ubiquitination, H3 K4 and K36 methylation, RNA pol II S5 and S2 phosphorylation and PAFc recruitment. These findings suggest that multiple interactions between RNMT-RAM, RNA pol II factors and RNA along the transcription unit stimulate transcription.
2018-05-01 | GSE87767 | GEO
Project description:An acute protein depletion system coupled with proteomic and genomic analyses reveal how SPT5 regulates promoter-proximal Pol II stability in eukaryotic cells, linking transcriptional initiation to productive elongation.
2021-02-04 | MSV000086806 | MassIVE
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:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.
2021-07-15 | PXD024457 | Pride
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:Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3’ mRNA isoforms for most genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.
2020-09-01 | GSE151196 | GEO
Project description:Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet mechanistic details of this evolution remain elusive. By comparing the transcriptional landscape of distantly related budding and fission yeast, we catch a glimpse of divergence in gene regulation. Using an adapted Precision Run-On sequencing (PRO-seq) approach, we have mapped the positions of RNA polymerase (RNA Pol II) active sites genome-wide in S. pombe and S. cerevisiae at base-pair resolution. We further mapped preferred sites of transcription initiation in each organism, exposing the origins of nascent transcription. Performing PRO-seq in strains lacking Spt4, a highly conserved elongation factor subunit, results in globally elevated levels of transcribing Pol II within genes in both species. Messenger RNA abundance, however, does not reflect the increases in RNA Pol II density, indicating a possible global elongation rate defect. Unexpectedly, we identify a novel pause in early elongation, specific to S. pombe, which requires Spt4, suggesting regulatory potential similar to promoter-proximal pausing in mammals.
2016-04-16 | GSE76142 | GEO