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 Citations (Scopus)

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

Fingerprint

mRNA guanylyltransferase
Caenorhabditis elegans
Messenger RNA
Enzymes
Protein Tyrosine Phosphatases
Complementary DNA
RNA
Mutagenesis
RNA Polymerase II
Cloning
Essential Genes
RNA Interference
Gene expression
Yeast
Saccharomyces cerevisiae
Protons
Organism Cloning
Elongation
Embryonic Structures
Nucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)
  • Molecular Biology
  • Cell Biology

Cite this

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

The Caenorhabditis elegans mRNA 5′-capping enzyme : In vitro and in vivo characterization. / Takagi, Toshimitsu; Walker, Amy K.; Sawa, Chika; Diehn, Felix; Takase, Yasutaka; Blackwell, T. Keith; Buratowski, Stephen.

In: Journal of Biological Chemistry, Vol. 278, No. 16, 18.04.2003, p. 14174-14184.

Research output: Contribution to journalArticle

Takagi, T, Walker, AK, Sawa, C, Diehn, F, Takase, Y, Blackwell, TK & Buratowski, S 2003, 'The Caenorhabditis elegans mRNA 5′-capping enzyme: In vitro and in vivo characterization', Journal of Biological Chemistry, vol. 278, no. 16, pp. 14174-14184. https://doi.org/10.1074/jbc.M212101200
Takagi, Toshimitsu ; Walker, Amy K. ; Sawa, Chika ; Diehn, Felix ; Takase, Yasutaka ; Blackwell, T. Keith ; Buratowski, Stephen. / The Caenorhabditis elegans mRNA 5′-capping enzyme : In vitro and in vivo characterization. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 16. pp. 14174-14184.
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