title: Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control.
abstract: RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Pei
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002455:Cell Division; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000378:metabolism*; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D016213:Cyclins; D004268:DNA-Binding Proteins; C105853:Pch1 protein, S pombe; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D035602:Transcriptional Elongation Factors; C087021:res2 protein, S pombe; C069940:SPT5 transcriptional elongation factor; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases