Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

• Published in: The Journal of biological chemistry Vol. 278; no. 9; pp. 7180 - 7188
• Main Authors: Pei, Yi, Schwer, Beate, Shuman, Stewart
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 28.02.2003
• Subjects: Amino Acid Sequence; Baculoviridae - genetics; Binding Sites; Cell Division; Chromatography, Affinity; Chromosomal Proteins, Non-Histone; Cyclin-Dependent Kinase 9; Cyclin-Dependent Kinases - chemistry; Cyclin-Dependent Kinases - metabolism; Cyclins - chemistry; Cyclins - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Gene Deletion; Models, Biological; Molecular Sequence Data; Mutation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; RNA, Messenger - metabolism; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - metabolism; Sequence Homology, Amino Acid; Transcription, Genetic; Transcriptional Elongation Factors - metabolism; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M211713200

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.