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...
Reovirus sigma3 is a virion outer shell protein that also binds dsRNA and stimulates translation by blocking activation of the dsRNA-dependent protein kinase, PKR. Purified sigma3 was shown by gel shift assay to bind specifically to RNA duplexes of minimal length 32-45 base pairs. PKR binding to dsRNA was prevented by sigma3, and translation inhibition of luciferase reporter by PKR expression in transfected cells was reversed by sigma3. Association of sigma3 with its outer capsid partner mu1/mu1C eliminated dsRNA binding and prevented restoration of protein synthesis. Analyses of sigma3 mutants demonstrated a direct correlation between dsRNA binding and reversal of the down-regulation of translation by PKR. In infected cells, sigma3 was stable but dsRNA binding decreased, presumably due to mu1/mu1C complex formation. The results suggest a functional transition from early inhibition of PKR activation by sigma3 to its association with mu1/mu1C in capsid structures.
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