title: Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.
abstract: Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control.
authors.affiliation: From the ‡Department of Internal Medicine; §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences;
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.affiliation: From the ‡Department of Internal Medicine; §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences;
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences;
authors.forename: Mridul
authors.initials: M
authors.lastname: Kalita
authors.affiliation: ¶Institute for Translational Sciences; ‖Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas.
authors.forename: Xueling
authors.initials: X
authors.lastname: Li
authors.affiliation: From the ‡Department of Internal Medicine; ¶Institute for Translational Sciences;
authors.forename: Mohammad
authors.initials: M
authors.lastname: Jamaluddin
authors.affiliation: From the ‡Department of Internal Medicine; §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences;
authors.forename: Bing
authors.initials: B
authors.lastname: Tian
authors.affiliation: From the ‡Department of Internal Medicine;
authors.forename: Chukwudi B
authors.initials: CB
authors.lastname: Edeh
authors.affiliation: §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences; ‖Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas.
authors.forename: John E
authors.initials: JE
authors.lastname: Wiktorowicz
authors.affiliation: §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences; ‖Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Kudlicki
authors.affiliation: From the ‡Department of Internal Medicine; §Sealy Center for Molecular Medicine; ¶Institute for Translational Sciences; arbrasie@utmb.edu.
authors.forename: Allan R
authors.initials: AR
authors.lastname: Brasier
mesh_terms: D045744:Cell Line, Tumor; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D025941:Protein Interaction Mapping; D012326:RNA Splicing; D014158:Transcription, Genetic
chemical_list: C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; C102004:DDX17 protein, human; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases