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

The transcription cycle of RNA polymerase II(Pol II) is regulated at discrete transition pointsby cyclin-dependent kinases(CDKs). Positive transcription elongation factorb ( P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanismsand targets of Cdk9 action remain largely unknown. Here, by a chemical geneticstrategy, 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” exoribonucleaseXrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFbsubstrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimeticsubstitution 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-TEFbregulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.