ISOLATION AND CHARACTERIZATION OF A HUMAN CDNA FOR MESSENGER-RNA 5'-CAPPING ENZYME

The human mRNA 5'-capping enzyme cDNA was identified. Three highly rel ated cDNAs, HCE1 (human mRNA capping enzyme 1), HCE1A and HCE1B, were isolated from a HeLa cDNA library. The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein. A short region of 69 bp in the 3'-h alf of the HCE1 ORF was missing in HCE1A and HCE1B, and, additionally, HCE1B has an early translation termination signal, which suggests tha t the latter two cDNAs represent alternatively spliced product. When e xpressed in Escherichia coil as a fusion protein with glutathione S-tr ansferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRN A 5'-guanylyltransferase (GTase) activities, and it formed a cap struc ture at the 5'-triphosphate end of RNA, demonstrating that it indeed s pecifies an active mRNA 5'-capping enzyme. The recombinant proteins de rived from HCE1A and HCE1B possessed only TPase activity. When express ed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Sacch aromyces cerevisiae CEG1 and CET1, the genes for GTase and TPase, resp ectively. These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity. In addition, the human TPase domain cannot functi onally substitute for the yeast enzyme in vivo.