P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

Abstract

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5 ′ -to-3 ′ "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics

“…In comparison with the ~170 potential CDK9 kinase substrates recently identified in HCT116 cells (Sanso et al, 2016), it is notable that the high-confidence substrates for CDK9 are distinct from the Mediator kinases. This further suggests that CDK9 (e.g.…”

CDK8 Kinase Activity Promotes Glycolysis

“…To address this knowledge gap, we engineered HCT116 CRC cells to express CDK8-AS that is sensitive to inhibition by non-hydrolysable ATP analogs. Mutation of a conserved phenylalanine ‘gatekeeper’ residue in kinase active sites (F97 in CDK8) to glycine creates ‘analog-sensitive’ kinases able to accept bulky ATP analogs such as 3MB-PP1 (Figure 1A) (Blethrow et al, 2004), an approach that has been used to dissect the function of other transcriptional CDKs (Larochelle et al, 2006; Larochelle et al, 2007; Sanso et al, 2016). We utilized a two-round genome editing strategy that combined transcription activator-like effector nuclease (TALEN)-mediated double-strand break generation with a recombinant adeno-associated virus (rAAV)-based homology donor construct to sequentially introduce the F97G mutation at both CDK8 alleles (Figure 1B and S1A) (see also Experimental Procedures).…”

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

“…To further assess P-TEFb signaling in neonatal megakaryopoiesis, additional downstream targets were examined. One such target, P-TEFb-mediated RNAPII CTD phosphorylation, is best reflected by the relative abundance of the more slowly migrating II0 isoform (18,19). RNAPII phosphorylation on S2 does not provide a reliable readout, as CDK9 phosphorylates multiple positions on the CTD and multiple kinases phosphorylate the S2 position (20)(21)(22)(23).…”