Publications

• Cloning and characterization of three human cDNAs encoding mRNA (guanine-7-)methyltransferase, an mRNA cap methylase.

  Tsukamoto T., Shibagaki Y., Niikura Y., Kiyohisa M.

  The mRNA cap structure is synthesized by a series of reactions catalyzed by capping enzyme and mRNA (guanine-7-)-methyltransferase. mRNA (guanine-7-)-methyltransferase catalyzes the methylation of GpppN-at the guanine N7 position, which is an essential step for gene expression in eukaryotic cells. ... >> More

  The mRNA cap structure is synthesized by a series of reactions catalyzed by capping enzyme and mRNA (guanine-7-)-methyltransferase. mRNA (guanine-7-)-methyltransferase catalyzes the methylation of GpppN-at the guanine N7 position, which is an essential step for gene expression in eukaryotic cells. Here we isolated three human cDNAs encoding mRNA (guanine-7-)-methyltransferase termed hCMT1a, hCMT1b and hCMT1c. hCMT1a and hCMT1b encode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. The third cDNA hCMT1c seems to encode the same polypeptide as hCMT1a, however, the 3'-noncoding region of hCMT1c contains sequences corresponding to part of the C-terminal coding and noncoding regions of hCMT1b thus consisting of a mosaic of hCMT1a and hCMT1b. RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)-methyltransferases. The recombinant hCMT1a expressed in E. coli exhibited mRNA (guanine-7-)-methyltransferase activity. On the other hand, neither mRNA (guanine-7-)-methyltransferase nor mRNA (nucleoside-2'-O-)-methyltransferase activity was detected with the recombinant hCMT1b protein. Although the biological significance of the expression of these three mRNA (guanine-7-)-methyltransferase mRNA species remains unknown at present, the nucleotide sequences suggest that they are produced by alternative RNA splicing. << Less

  Biochem. Biophys. Res. Commun. 251:27-34(1998) [PubMed] [EuropePMC]

• Modification of the 5'-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus.

  Ensinger M.J., Martin S.A., Paoletti E., Moss B.

  RNA guanylyl and methyl transferases have been solubilized from vaccinia virus cores. The guanylyl transferase specifically adds a GMP residue to the 5'-terminus of unmethylated vaccinia virus mRNA to form the structures G(5')ppp(5')Gp- and G(5')ppp(5')Ap-. Studies with [alpha-32P]GTP and [beta, g ... >> More

  RNA guanylyl and methyl transferases have been solubilized from vaccinia virus cores. The guanylyl transferase specifically adds a GMP residue to the 5'-terminus of unmethylated vaccinia virus mRNA to form the structures G(5')ppp(5')Gp- and G(5')ppp(5')Ap-. Studies with [alpha-32P]GTP and [beta, gamma-32P]GTP indicated that only the alpha-phosphate is transferred. In the presence of S-adenosylmethionine, the methyl transferases convert the blocked 5'-termini to m7G(5')ppp(5')Gmp- and m7G(5')ppp(5')Amp-. Similarly, the enzymes can modify synthetic poly(A) to form the structure m7G(5')ppp(5')Amp-. << Less

  Proc Natl Acad Sci U S A 72:2525-2529(1975) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Recombinant human mRNA cap methyltransferase binds capping enzyme/RNA polymerase IIo complexes.

  Pillutla R.C., Yue Z., Maldonado E., Shatkin A.J.

  Guanine N-7 methylation is an essential step in the formation of the m7GpppN cap structure that is characteristic of eukaryotic mRNA 5' ends. The terminal 7-methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression including mRNA processing, transport, and ... >> More

  Guanine N-7 methylation is an essential step in the formation of the m7GpppN cap structure that is characteristic of eukaryotic mRNA 5' ends. The terminal 7-methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression including mRNA processing, transport, and translation. Here we describe the cloning, primary structure, and properties of human RNA (guanine-7-)methyltransferase. Sequence alignment of the 476-amino acid human protein with the corresponding yeast ABD1 enzyme demonstrated the presence of several conserved motifs known to be required for methyltransferase activity. We also identified a Drosophila open reading frame that encodes a putative RNA (guanine-7-)methyltransferase and contains these motifs. Recombinant human methyltransferase transferred a methyl group from S-adenosylmethionine to GpppG 5'ends, which are formed on RNA polymerase II transcripts by the sequential action of RNA 5'-triphosphatase and guanylyltransferase activities in the bifunctional mammalian capping enzyme. Binding studies demonstrated that the human cap methyltransferase associated with recombinant capping enzyme. Consistent with selective capping of RNA polymerase II transcripts, methyltransferase also formed ternary complexes with capping enzyme and the elongating form of RNA polymerase II. << Less

  J. Biol. Chem. 273:21443-21446(1998) [PubMed] [EuropePMC]

• Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

  Martin S.A., Moss B.

  A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA gu ... >> More

  A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG-are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article). << Less

  J Biol Chem 250:9330-9335(1975) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates.

  Groner Y., Gilboa E., Aviv H.

  HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of G ... >> More

  HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm-capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains. << Less

  Biochemistry 17:977-982(1978) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

  Mao X., Schwer B., Shuman S.

  RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated pol ... >> More

  RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo. << Less

  Mol. Cell. Biol. 15:4167-4174(1995) [PubMed] [EuropePMC]

• Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

  Martin S.A., Paoletti E., Moss B.

  The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guan ... >> More

  The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system. << Less

  J Biol Chem 250:9322-9329(1975) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.