Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Abstract

ABSTRACT5-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5-triphosphatase activity and catalyzed formation of RNA 5-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.Addition of a 5Ј-terminal cap is an important, early event in mRNA formation (1). This structural hallmark of most eukaryotic mRNAs enhances splicing (2-4), transport (5), translation (6), and stability (7,8) and is essential for viability (9).Caps are formed on nascent nuclear pre-mRNAs by conversion of 5Ј-tri-diphosphate to 5Ј-diphosphate ends, followed by addition of GMP and methylation (1, 10). The guanylyltransfer reaction characterized in various systems involves formation of an active enzyme intermediate containing GMP covalently attached to lysine (11). In yeast, mRNA capping enzyme consists of separate subunits for RNA 5Ј-triphosphatase and guanylyltransferase activities (9, 12). cDNA clones coding for mRNA guanylyltransferase in Saccharomyces cerevisiae (9), Schizosaccharomyces pombe (13), and Candida albicans (14) have been sequenced. Each contains the active site lysine in KXDG (13, 15), one of several highly conserved motifs characteristic of a superfamily of nucleotidyl transferases (16). A number of viral capping enzymes also contain these diagnostic sequence motifs, and the recently solved structure of capping enzyme from Chlorella virus PBCV-1 suggests that specific residues in these motifs are important for binding GTP (17). Despite this detail of sequence and structure information, no metazoan capping enzyme previously has been cloned and characterized.To explore the molecular interactions that result in selective capping of RNA polymerase II (pol II) transcripts in mammalian cells, we have isolated and characterized cDNA clones that code for the human and mouse capping enzymes. Functional studies demonstrated that the mammalian enzyme complements the lethality of a S. cerevisiae mu...

Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

“…Recent studies have also shown that pre-mRNAs are targeted for capping through binding of the guanylyltransferase component of the capping apparatus to the phosphorylated CTD of Pol II (6,33,72). Mammalian guanylyltransferase binds synthetic CTD peptides containing phosphoserine at either Ser-2 or Ser-5 of the YSPTSPS repeat (16).…”

Modulation of TFIIH‐associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II

“…In fact, it has been reported that PolII possessing the hyperphosphorylated CTD of the largest subunit (IIo form) does not participate in the transcription PIC (Lu et al 1991), while actively transcribing PolII contains the IIo form of the largest subunit (Weeks et al 1993;O'Brien et al 1994). Recently, it was reported that the phosphorylated CTD has a role in recruiting the mRNA capping enzyme to the nascent transcript and that mRNA capping occurs soon after promoter clearance (Coppola et al 1983;Cho et al 1997;McCracken et al 1997a,b;Yue et al 1997). These results suggest that CTD phosphorylation occurs during transcription initiation and promoter clearance.…”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…The earliest processing step is mRNA capping, which can occur as soon as the 59 triphosphate terminus of the nascent RNA extrudes from the elongating RNA polymerase (Chiu et al 2002). The capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II McCracken et al 1997;Yue et al 1997;Ho and Shuman 1999;Pei et al 2001;Fabrega et al 2003). Capping enzymes also bind to the Pol II elongation factor Spt5 (Wen and Shatkin 1999;Pei and Shuman 2002), which, in conjunction with its binding partner Spt4, elicits an elongation arrest at promoter-proximal sites (Wada et al 1998a).…”