Dataset Information

Identification of CDK9 targets using DRB and quantitative phosphoproteomics

ABSTRACT: CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Michael Tellier

LAB HEAD: Shona Murphy

PROVIDER: PXD026720 | Pride | 2022-08-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Oxidation__M_Sites.txt	Txt
	Phospho__STY_Sites.txt	Txt
	Qex01_SVH_170316_MichaelTellier_1_4_10_013.raw	Raw
	Qex01_SVH_170316_MichaelTellier_1_4_11_015.raw	Raw
	Qex01_SVH_170316_MichaelTellier_1_4_12_016.raw	Raw

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Publications

Short communication: Effect of washing method, grinding size, and the determination of an indigestible fraction on in situ degradation of starch in mature corn grain.

Fernandes T T Ávila C L S CLS Pereira M N MN Ferraretto L F LF

Journal of dairy science 20180809 10

The objectives of this study were to determine (1) the effect of grinding size (1, 2, 4, and 6 mm) to determine effective ruminal disappearance (ERD); (2) the most adequate method to estimate the rapidly degradable fraction (A); (3) a time point to measure the indigestible fraction (C); and (4) the viability of using fewer time points to estimate starch fractional disappearance rate (k<sub>d</sub>) of mature corn grain. Fraction A was determined by rinsing in a bucket or washing machine, rumen i ...[more]

PMID: 30100508

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Project description:RNA Polymerase II (Pol II)-dependent transcription is meticulously regulated during initiation, elongation, termination, and two pivotal transitions: from initiation to elongation and from elongation to termination 1. The successful transition from elongation to termination is vital for accurate transcription termination 2,3. While elongation and termination have been extensively studied, the regulatory mechanisms governing the transition between these stages are not fully understood. Here we report that, in fission yeast Schizosaccharomyces pombe (S. pombe) and human cells, Cdk9 and its cyclin partner (Cyclin T in humans, and Pch1 in S. pombe) disengage from the elongation machinery near gene 3'-ends, as Pol II crosses the cleavage and polyadenylation signal (CPS). Concurrently, chromatin immunoprecipitation (ChIP) data shows an increased association of Dis2 (the PP1 ortholog in S. pombe)4 beyond the CPS. Additionally, ChIP-seq indicates that histone 2B monoubiquitylation (H2Bub1) is pivotal for Cdk9 occupancy but serves differential roles along the gene. H2Bub1 facilitates Cdk9 chromatin recruitment during elongation but prompts Cdk9 dissociation from the elongation complex as Pol II traverses the CPS. Furthermore, in S. pombe, disruptions of H2Bub1 in htb1-K119R 5 and sustained levels in ubp8D 6 inversely affect the chromatin occupancy of Pol II, Pol II CTD phosphorylation within the heptad repeats, Spt5, phosphorylated Spt5 (pSpt5), Dis2, components of the mRNA 3’-end processing complex (Pfs2 and Pla1), and termination factors Rhn1 (Rtt103) and Pcf1 around the CPS—diminishing with H2Bub1 loss and increasing with H2Bub1 stabilization. In essence, our data indicates that the H2Bub1-mediated eviction of Cdk9 as Pol II navigates the CPS precipitates a rise in PP1 binding, which, in turn, dephosphorylates pSpt5, slowing Pol II to facilitate efficient termination. The unique modulation of Cdk9 occupancy by H2Bub1 during transcription progression presents a compelling, novel concept requiring further exploration.