The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization.
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.
Takagi
Toshimitsu
T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walker
Amy K
AK
Sawa
Chika
C
Diehn
Felix
F
Takase
Yasutaka
Y
Blackwell
T Keith
TK
Buratowski
Stephen
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D020224:Expressed Sequence Tags; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D016366:Open Reading Frames; D010641:Phenotype; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D000098556:mRNA Guanylyltransferases
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases