The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

J Biol Chem. 2003 Apr 18;278(16):14174-84. doi: 10.1074/jbc.M212101200. Epub 2003 Feb 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Caenorhabditis elegans / metabolism*
  • Cell Nucleus / metabolism
  • Cloning, Molecular
  • DNA, Complementary / metabolism
  • Databases as Topic
  • Dose-Response Relationship, Drug
  • Expressed Sequence Tags
  • Gene Expression Regulation*
  • Genetic Complementation Test
  • Humans
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Mice
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / genetics*
  • Nucleotidyltransferases / physiology*
  • Open Reading Frames
  • Phenotype
  • Protein Structure, Tertiary
  • RNA Interference
  • RNA, Messenger / metabolism
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Sequence Homology, Amino Acid

Substances

  • DNA, Complementary
  • RNA, Messenger
  • Recombinant Proteins
  • Nucleotidyltransferases
  • mRNA guanylyltransferase