Origins of mRNA identity: capping enzymes bind to the phosphorylated C-terminal domain of RNA polymerase II.

Cellular enzymes that cap, splice, and polyadenylate eukaryotic pre-mRNAs are targeted in vivo to the nascent chains synthesized by RNA polymerase II (pol II). Placing a mammalian pol II transcription unit under the control of a pol III promoter results in a failure to cap, splice, or polyadenylate the transcript (1, 2). How is pre-mRNA ‘‘identity’’ established? Do the various mRNA processing enzymes recognize protein components of the pol II transcription elongation complex or is identity conferred through an initial pol II-specific modification that directs subsequent mRNA fate? We know that acquisition of the m7GpppN cap is the first modification event in mRNA biogenesis and that capping facilitates downstream transactions such as splicing, polyadenylation, transport, and translation. What we don’t understand is how pol II transcripts are specifically singled out for capping. Now, three groups of investigators (the Shatkin and Reinberg laboratories collaboratively, the Bentley and Shuman laboratories collaboratively, and the Buratowski laboratory) have presented findings that offer an elegant solution to the puzzle: the capping enzymes are targeted to pre-mRNA by binding to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA pol II (3–5). In the paper by Yue et al. (3) in this issue of the Proceedings, the Shatkin and Reinberg group show that mammalian mRNA capping enzyme interacts directly with pol IIO, the hyperphosphorylated form of pol II, but not with pol IIA, the form in which the CTD is either unphosphorylated or hypophosphorylated. The CTD, which is unique to pol II, consists of a tandem array of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The mammalian pol II large subunit has 52 tandem repeats, whereas the Saccharomyces cerevisiae subunit has 27 copies. The IIA and IIO forms of pol II are interconvertible and functionally distinct. In vivo, the pol IIO enzyme contains as many as 50 phosphorylated amino acids (primarily phosphoserine) within the CTD (6). During transcription initiation, pol IIA is recruited to the DNA template by the general transcription factors TBP (TATAbinding protein), TFIIB (transcription factor IIB), and TFIIF. TBP has been reported to bind to pol IIA, but not pol IIO (7). The pol IIA CTD undergoes extensive phosphorylation and conversion to IIO during the transition from preinitiation complex to stable elongation complex. The CTD kinase activity of transcription factor IIH is implicated in CTD hyperphosphorylation during this step (8). TFIIH contains a cyclin and cyclin-dependent kinase subunit pair (cdk7 and cyclin H) that catalyzes phosphorylation of Ser-5 of the CTD heptapeptide (9). TFIIH is recruited to the preinitiation complex by TFIIE, which binds specifically to pol IIA (10). TFIIE and TFIIH dissociate from the transcription complex shortly after initiation (11). Capping occurs when nascent RNA chains grow to '30 nucleotides in length, at which point their 59 ends are extruded from the RNA binding pocket of the polymerase and are thereby accessible to the capping enzymes (12, 13). Cap formation entails a series of three reactions catalyzed by RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase. The triphosphatase hydrolyzes the 59 triphosphate end of the primary transcript to a diphosphate. Guanylyltransferase adds GMP from GTP to the diphosphate RNA end to form a blocked G(59)ppp(59)N structure. The methyltransferase adds a methyl group from S-adenosylmethionine to the cap guanosine to form m7GpppN. This pathway was elucidated more than 20 years ago by the Moss and Shatkin laboratories through studies of the capping enzymes encoded by vaccinia virus and reovirus (reviewed in ref. 14). Only in the past few years have cellular genes encoding the capping enzymes been cloned (15–19). The fact that the cap guanylyltransferase and cap methyltransferase activities are both essential for yeast cell growth underscores the critical role of the cap in mRNA metabolism. In their paper, Yue et al. (3) identify cDNAs encoding the human and mouse guanylyltransferases—the first examples of cloned capping enzymes from mammals. The mouse and human capping enzymes are 597-aa polypeptides with 95% amino acid sequence identity. Like the 573-aa Caenorhabditis elegans capping enzyme (18, 19), the mammalian proteins are bifunctional and consist of an N-terminal RNA triphosphatase domain linked to a C-terminal guanylyltransferase domain. The metazoan guanylyltransferase domains are structurally similar to the monofunctional guanylyltransferase proteins encoded by yeast and Chlorella virus and to the guanylyltransferase domain of vaccinia capping enzyme. The N-terminal triphosphatase domain contains the signature motif of the protein tyrosine phosphatase superfamily. The authors provide clear biochemical evidence that the mouse cDNA encodes a catalytically active triphosphatase-guanylyltransferase. Moreover, they find that the mouse cDNA rescues growth of a yeast strain lacking the endogenous guanylyltransferase gene (20). To examine the polymerase-capping connection, Yue et al. (3) incubated mammalian capping enzyme with partially purified RNA pol II consisting of a mixture of the IIO and IIA isoforms, and then immunoprecipitated the sample with antibody to the guanylyltransferase domain. Analysis of the precipitate by Western blotting using an antibody against the largest subunit of RNA pol II revealed that pol IIO, but not pol IIA, was precipitated by the capping enzyme antibody. The guanylyltransferase domain alone was sufficient for selective binding of pol IIO. This simple experiment has broad implications. Does the capping enzyme interact directly with the CTD? McCracken et al. (4) have shown by CTD affinity chromatography that mammalian guanylyltransferase binds to a recombinant glutathione-S-transferase–CTD fusion protein containing 15 tandem copies of the heptapeptide, provided that the CTD has been phosphorylated in vitro by HeLa cell extract, recombinant cdk7ycyclin H kinase, or cdc2 kinase. The guanylyltransferase does not bind to nonphosphorylated CTD or to in vitro phosphorylated mutant CTD in which residue Ser-5 of each heptapeptide repeat was replaced by alanine. This engenders a model whereby phosphorylation of Ser-5 suffices © 1997 by The National Academy of Sciences 0027-8424y97y9412758-3$2.00y0 PNAS is available online at http:yywww.pnas.org.