title: 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.
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;
authors.forename: Miriam
authors.initials: M
authors.lastname: Sansó
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94143, USA; Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143, USA;
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Levin
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94143, USA; Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143, USA;
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Lipp
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA;
authors.forename: Vivien Ya-Fan
authors.initials: VY
authors.lastname: Wang
authors.affiliation: Department of Structural Immunology, Institute of Innate Immunity, University of Bonn, 53127 Bonn, Germany;
authors.forename: Ann Katrin
authors.initials: AK
authors.lastname: Greifenberg
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Quezada
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;
authors.forename: Akbar
authors.initials: A
authors.lastname: Ali
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;
authors.forename: Animesh
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;
authors.forename: Stéphane
authors.initials: S
authors.lastname: Larochelle
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA; Department of Pediatrics, University of California at San Diego School of Medicine, La Jolla, California 92093, USA.
authors.forename: Tariq M
authors.initials: TM
authors.lastname: Rana
authors.affiliation: Department of Pediatrics, University of California at San Diego School of Medicine, La Jolla, California 92093, USA.
authors.forename: Matthias
authors.initials: M
authors.lastname: Geyer
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA;
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94143, USA; Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143, USA;
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;
authors.forename: Robert P
authors.initials: RP
authors.lastname: Fisher
mesh_terms: D002843:Chromatin / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D005820:Genetic Testing; D045325:HCT116 Cells; D006801:Humans; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D002843:Chromatin; D042925:Positive Transcriptional Elongation Factor B; D042863:Cyclin-Dependent Kinase 9; D005095:Exoribonucleases; C120903:XRN2 protein, human
keywords: P-TEFb; RNA polymerase II; Xrn2; chemical genetics; protein phosphorylation; transcription termination