The transcriptional elongation rate regulates alternative polyadenylation in yeast
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PROVIDER: PRJNA635106 | ENA |
REPOSITORIES: ENA
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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:RNA polymerase II elongation rate controls gene expression by alternative polyadenylation
| PRJNA590527 | ENA
Project description:Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ UTRs. Profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. In yeast, upstream and downstream shifts within isoform clusters occur steadily at the nucleotide level. In contrast, changes from one isoform to the next are much smaller between clusters, even when the distances between them are relatively large. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.
2022-11-28 | GSE214095 | GEO
Project description:To study the impact of the RNA polymerase II (Pol II) elongation rate on gene expression, we used CRISPR-Cas9 genome editing in S. pombe to generate a "slow" Pol II mutant with decreased elongation rate. Although the mutation is well tolerated as far as cell growth is concerned, transcriptomic analyses revealed that the slow mutant tends to terminate transcription prematurely. We distinguished two mechanisms by which premature termination affects gene expression in the slow mutant: It either (1) shortens 3'UTR, or (2) derepresses protein coding genes by prematurely terminating upstream interfering RNAs. Strikingly, the genes affected by these mechanisms are enriched for genes involved in phosphate uptake and purine synthesis, two processes essential for the maintenance of the nucleotide pool of the cell. Together with evidences that nucleotides are conditional for Pol II processive elongation, our results suggest that Pol II elongation rate acts as both sensor and effector in response to nucleotide depletion.
2020-05-29 | GSE140698 | GEO
Project description: Not available
| S-EPMC7532003 | biostudies-literature
Project description:Yeast mRNAs are polyadenylated at multiple sites in their 3’ untranslated regions (3’UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the relationship between Pol II pause sites and poly(A) sites within 3’UTRs.
2023-08-28 | GSE234406 | GEO
Project description:Alternative cleavage and polyadenylation (APA) is a mechanism generating multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here we used 3' Region Extraction And Deep Sequencing (3'READS) to systematically map cleavage and polyadenylation sites (PAS) in Drosophila and analyzsed APA in the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate. We found that about two thirds of mRNA genes in fly undergo APA and cis element analysis indicates that fly PASs are associated with downstream UGUG elements to a lesser degree than mammalian PASs. The head tissue tends to use distal PASs compared to the body, leading to preferential expression of long 3'UTR isoforms and transcripts using distal terminal exons. Gene Ontology analysis correlates brain-specific APA events with neuronal functions, indicating their functional relevance. We also found that the distance between the proximal and distal PASs and intron locations might play important roles for brain APA. Analysis of the RpII215 C4 mutant strain, reveals that a 50% decrease in transcriptional elongation rate leads to shortened 3'UTRs in the fly body, but not in the head. This correlates with an upregulation of Ssu72 specifically in RpII215 C4 bodies, suggesting that the mechanism underlying those observations involves another layer of regulation by RNAPII termination factors. By contrast, more significant activation of intronic PASs was observed in the head compared to the body when RNAPII speed is slow. Together, our results reveal that in Drosophila RNAPII elongation impacts on PAS choice in a context-specific manner.
2017-08-23 | GSE85368 | GEO
Project description:Divergence of alternative polyadenylation in mouse
| PRJEB15336 | ENA
Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3â ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells
2016-01-15 | E-GEOD-72574 | biostudies-arrayexpress
Project description:Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects. HeLa cell line was stably transfected with shRNA plasmids targeting CstF64. Total RNA was isolated from CstF64 KD cells and wild-type control cells using Trizol according to manufacturerâs protocols. Samples were deep sequenced in duplicate using the Illumina GAIIx system.
2016-03-14 | E-GEOD-79157 | biostudies-arrayexpress