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 Nterminal 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.The 5Ј-cap is the defining structural feature of eukaryotic mRNA. Consisting of m 7 G linked via an inverted 5Ј-5Ј triphosphate bridge to the initiating nucleoside of the transcript, the cap is formed by enzymatic modification of pre-mRNAs as they are being synthesized by RNA polymerase II (pol II). 1 Capping entails three reactions: (i) the 5Ј-triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase, (ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase, and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (1). Targeting of cap formation to pre-mRNAs depends on interactions of the capping enzymes with the phosphorylated C-terminal domain (CTD) of the largest subunit of pol II (Ref. 2 and citations therein). Recruitment of the capping apparatus to the elongation complex requires the TFIIH-associated CTD kinase (Kin28 in yeast, Cdk7 in mammals), which phosphorylates Ser-5 of the CTD heptad repeat YSPTSPS (3, 4).Other protein-protein contacts may also be involved in coupling capping to pol II transcription elongation. The pol II elongation factor Spt5 binds directly to the triphosphatase and guanylyltransferase components of the mammalian and Schizosaccharomyces pombe capping apparatus (5, 6). The fission yeast S. pombe employs a distinctive strategy of cap targeting whereby the triphosphatase (Pct1) and guanylyltrans...

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

“…The capacity of SpCdk9/Pch1 to phosphorylate the CTD arrays of both pol II and Spt5 in vitro echoed the substrate specificity of metazoan P-TEFb (11,12,31,32). These findings suggested 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 via phosphorylation of one or more components of the pol II elongation complex (30).…”

“…Complementation of the S. cerevisiae bur1⌬ and bur2⌬ mutants by coexpression of SpCdk9 and Pch1 showed that the fission yeast proteins are genuine orthologs of Bur1/Bur2, a putative fungal P-TEFb. Analysis of the recombinant SpCdk9/Pch1 complex produced in baculovirus-infected insect cells showed that the S. pombe proteins comprise a bona fide protein kinase with a heterodimeric quaternary structure (30). The capacity of SpCdk9/Pch1 to phosphorylate the CTD arrays of both pol II and Spt5 in vitro echoed the substrate specificity of metazoan P-TEFb (11,12,31,32).…”

“…Site-directed Mutagenesis of SpCdk9 -Plasmid pYX-SpCDK9 (CEN TRP1) contains the cDNA encoding full-length wild-type SpCdk9 under the control of the S. cerevisiae TPI1 promoter (30). Amino acid substitution mutations were introduced into the SpCDK9 gene by two-stage overlap extension PCR.…”

“…Protein Kinase Assay-The SpCdk9/His-Pch1 kinase complex was isolated from Sf9 insect cells coinfected with recombinant baculoviruses expressing SpCdk9 or His-Pch1 by nickel-nitrilotriacetic acid-agarose affinity chromatography as described previously (30). Kinase reaction mixtures (20 l) containing 50 mM Tris acetate (pH 6.0), 1 mM DTT, 2.5 mM MnCl 2 or 10 mM MgCl 2 , 50 M [␥-32 P]ATP, 4 g of GST-Spt5(801-990) containing the C-terminal nonapeptide repeat array of S. pombe Spt5, and SpCdk9/His-Pch1 were incubated for 60 min at 22°C.…”

“…1 The abbreviations used are: pol, polymerase; Cdk, cyclin-dependent kinase; CTD, carboxyl-terminal domain; DTT, dithiothreitol; 5-FOA, 5-fluoroorotic acid; GST, glutathione S-transferase; TEF, transcriptional elongation factor. homolog of metazoan Cdk9 and S. cerevisiae Bur1 (30). Initial studies of SpCdk9 included the identification of the essential S. pombe cyclin Pch1 as its binding partner.…”