N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5–phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

Production of messenger RNA in eukaryotic cells is a complex, multistep process. mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase. These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation. Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo. For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation. These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation. Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.

RNA polymerase II (RNAP II) is responsible for transcription of mRNA precursors in eukaryotic cells. Recent studies, however, have suggested that RNAP II also participates in subsequent RNA processing reactions through interactions between the carboxy-terminal domain (CTD) of the RNAP II largest subunit and processing factors. Using reconstituted in vitro splicing assays, we show that RNAP II functions directly in pre-mRNA splicing by influencing very early steps in assembly of the spliceosome. We demonstrate that the phosphorylation status of the CTD dramatically affects activity: Hyperphosphorylated RNAP IIO strongly activates splicing, whereas hypophosphorylated RNAP IIA can inhibit the reaction.Received March 3, 1999; revised version accepted April 6, 1999.Splicing of mammalian pre-mRNA is a nuclear process in which introns are removed from primary transcripts synthesized by RNA polymerase II (RNAP II). Splicing takes place in a large macromolecular complex called the spliceosome, which is composed of small nuclear ribonucleoprotein (snRNP) particles and non-snRNP proteins including members of the serine/arginine-rich (SR) protein family (for review, see Moore et al. 1993;Kramer 1996). Although cytological studies have suggested that splicing can occur cotranscriptionally (e.g., Beyer et al. 1988;Bauren and Wieslander 1994) and factors required for splicing are found localized at sites of active transcription (e.g., Zhang et al. 1994), functional coupling between transcription and splicing does not seem obligatory because splicing can be reconstituted in vitro with pretranscribed RNA and splicing-competent cell extracts.Recent studies, however, have provided evidence indicating that the carboxy-terminal domain (CTD) of the largest subunit of RNAP II links transcription with premRNA processing (for reviews, see Corden and Patturajan 1997;Neugebauer and Roth 1997;Steinmetz 1997). The CTD is comprised of multiple repeats of the consensus sequence YSPTSPS, which is highly conserved throughout evolution (for review, see Corden 1990) and subject to reversible phosphorylation during the transcription cycle (for review, see Dahmus 1996). RNAP II with a hypophosphorylated CTD (RNAP IIA) is preferentially included in the preinitiation complex at the promoter, whereas RNAP II with a hyperphosphorylated CTD (RNAP IIO) is associated with elongation complexes.Biochemical studies have shown that RNAP II, via the CTD, can physically interact with capping enzymes McCracken et al. 1997a;Yue et al. 1997), polyadenylation factors (McCracken et al. 1997b), and splicing factors, including both snRNPs and SR-like proteins (Chabot et al. 1995;Yuryev et al. 1996;Mortillaro et al. 1996;Kim et al. 1997). Notably, only RNAP IIO has been found to associate with capping and splicing factors, and this isoform has also been detected in active spliceosomes (Mortillaro et al. 1996). In addition, in vivo studies using mammalian cultured cells have demonstrated that RNAs transcribed by RNAP II with a shortened CTD undergo inefficient capping, ...

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays critical roles both in transcription of mRNA precursors and in the processing reactions needed to form mature mRNAs. The CTD undergoes dynamic changes in phosphorylation during the transcription cycle, and this plays a significant role in coordinating its multiple activities. But how these changes themselves are regulated is not well understood. Here we show that the peptidyl-prolyl isomerase Pin1 influences the phosphorylation status of the CTD in vitro by inhibiting the CTD phosphatase FCP1 and stimulating CTD phosphorylation by cdc2/cyclin B. This is reflected in vivo by accumulation of hypophosphorylated RNAP II in pin1 −/− cells, and of a novel hyper-hyperphosphorylated form in cells induced to overexpress Pin1. This hyper-hyperphosphorylated form of RNAP II also accumulates in M-phase cells, in a Pin1-dependent manner, and associates specifically with Pin1. Functionally, we find that Pin1 overexpression specifically inhibits ongoing transcription of mRNA precursors in vivo and both transcription and RNAP II-stimulated pre-mRNA splicing in cell extracts. Pin1 thus plays a significant role in regulating RNAP II CTD structure and function.

RNA polymerase II (Pol II) is the only polymerase to possess heptapeptide repeats in the C-terminal domain (CTD) of its large subunit. During transcription, CTD phopshorylation occurs and is maintained from initiation to termination. To date, among the three known CTD kinases possessing CDK-cyclin pairs, TFIIH is the only one that forms a preinitiation complex. The Mediator complex plays essential roles in transcription initiation and during the transition from initiation to elongation by transmitting signals from transcriptional activators to Pol II. P-TEFb specifically plays a role in transcription elongation. TFIIH and mediator phosphorylate serine 5 (Ser5) of the CTD heptapeptide repeat sequence, whereas P-TEFb phosphorylates serine 2 (Ser2). Recently, it has become clear that CTD phosphorylation is not only essential for transcription, but also as a platform for RNA processing and chromatin regulation. In this review, we discuss the central role of Pol II phosphorylation in these nuclear events.