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THOC5 complexes with DDX5, DDX17 and CDK12 to regulate R loop structures and transcription elongation rate

ABSTRACT: THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50-60% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence at the start site, on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites suggesting that THOC5 is involved in transcriptional elongation. Indeed, DRB/TTchem-seq that measures elongation rates in vivo revealed an accumulation of released Pol II near to TSS and a decrease of elongation rates in THOC5 depleted cells. Furthermore, THOC5 is more recruited to its target genes in cells expressing slow Pol II than those expressing fast Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. 3’ cleavage of 50% of THOC5 target genes is also regulated by CDK12 and THOC6. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE173374 | GEO | 2022/12/16

REPOSITORIES: GEO

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THOC5 complexes with other proteins (DDX5, DDX17 and CDK12) essential in primitive cell survival to regulate R loop structures and RNA elongation rate.

Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites except those at the promotor regions suggesting that THOC5 is involved in transcriptional elongation. Indeed, THOC5 independent genes in cells expressing fast Pol II became THOC5 dependent in cells expressing slow Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells

2022-12-12 | PXD035092 | Pride

A member of transcription/export complex (TREX), THOC5 controls elongation rate by recruiting CDK12.

Project description:THOC5, a member of the THO complex that is a subcomplex of Transcription/export complex (TREX) 1, is essential for 3´processing of slow kinetics IEGs, and for export of only a subset of genes. Applying nanopore mRNA-sequence technology, we show here that upon depletion of THOC5, 50% of mRNAs showed alteration of 3´end cleavage sites. Moreover, polymerase-II (Pol II)-CHIP-sequencing data reveals that upon depletion of THOC5 Pol II density was increased at gene body and 3´UTR. THOC5 is recruited near to Pol II high density sites except promotor regions, suggesting that THOC5 is involved in transcription elongation. Indeed, 385 THOC5 independent genes in fast Pol II cells became THOC5 dependent in slow Pol II cells. Chromatin associated THOC5 in slow Pol II cells interacts with CDK12, RNA helicases DDX5 and DDX, and THOC6, but not with other members of THO complex. Notably, THOC5 did not form this complex in fast poly II cells. CDK12 was recruited to R-loop (DNA:RNA hybrid) in THOC5/THOC6 dependent manner. Here, THOC6, but not THOC5 directly interacts with R-loop then, recruited DDX5 resolves R-loop and then CDK12 enhances further transcription elongation. These data show for the first time the novel function of THOC5/THOC6 complex during transcription elongation.

2022-12-31 | GSE166115 | GEO

The transcriptional elongation rate regulates alternative polyadenylation in yeast

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

DRB-PRO-Seq for measuring Pol II transcriptional elongation rate alterations upon inhibition of analog sensitive CDK12 and/or CDK13

Project description:We performed PRO-Seq to determine Pol II elongation rates by measruing the distance of Pol II travels during 25 minutes after Pol II being relased from DRB induced pausing. We allowed Pol II to release in the presence of 1-NM-PP1 or DMSO after DRB being wahsed out in order to assess the effects of CDK12 and/or CDK13 inhibtion on transcriptional elongation rates.

2020-04-29 | GSE141373 | GEO

Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase II termination suggest widespread kinetic competition

Project description:Transcription termination was analyzed by anti RNA pol II ChIP-seq in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates and in lines that inducbily over-express WT or an active site mutant of the RNA exonuclease "torpedo" Xrn2. Transcription termination zones were mapped by anti-pol II ChIP-seq under conditions where transcription elongation rate was increased or decreased by point mutations in the large subunit of the enzyme. Termination was also assayed under conditions where Xrn2 exonuclease activity was inhibited by over-expression of an active site mutant (D235A).

2015-11-19 | E-GEOD-72800 | biostudies-arrayexpress

Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase II termination suggest widespread kinetic competition

2015-11-19 | GSE72800 | GEO

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Project description:Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

2020-06-18 | GSE133156 | GEO

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

2020-06-03 | GSE115290 | GEO

Elongation rate of RNA polymerase II affects pausing patterns across the 3’ UTR

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

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