The Caenorhabditis elegans mRNA 5′-capping enzyme: In vitro and in vivo characterization

Toshimitsu Takagi, Amy K. Walker, Chika Sawa, Felix Diehn, Yasutaka Takase, T. Keith Blackwell, Stephen Buratowski

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.

Original languageEnglish (US)
Pages (from-to)14174-14184
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number16
DOIs
StatePublished - Apr 18 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Takagi, T., Walker, A. K., Sawa, C., Diehn, F., Takase, Y., Blackwell, T. K., & Buratowski, S. (2003). The Caenorhabditis elegans mRNA 5′-capping enzyme: In vitro and in vivo characterization. Journal of Biological Chemistry, 278(16), 14174-14184. https://doi.org/10.1074/jbc.M212101200