Publications

• 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.

• mRNA capping: biological functions and applications.

  Ramanathan A., Robb G.B., Chan S.H.

  The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap ... >> More

  The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose. << Less

  Nucleic Acids Res 44:7511-7526(2016) [PubMed] [EuropePMC]

  This publication is cited by 15 other entries.

• 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.

• 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.