Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control.
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.
Pei
Yi
Y
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Schwer
Beate
B
Shuman
Stewart
S
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
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