Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-.

Furuichi, Yasuhiro; Morgan, M.; Muthukrishnan, Subbaratnam; Shatkin, Aaron J.

doi:10.1073/pnas.72.1.362

Cited by 314 publications

(153 citation statements)

References 26 publications

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“…However, the wheat germ and virion methylase(s) both methylate the viral mRNA with great specificity. The 5' end is-the site of mRNA methylation in each case, and 5'-terminal, 3H-labeled 7-methylguanosine is obtained from VSV and reovirus mRNA methylated during transcription (7,12) The concomitant inhibition of viral mRNA methylation and translation by ATA is consistent with an interdependence of the two processes at the initiation step of polypeptide synthesis. The inhibitor blocks protein synthesis at the level of ribosome binding (19), suggesting that in wheat germ extracts the mRNA methylating activity may be ribosome-bound.…”

Section: Dependence Of Methylation On Initiation Of Protein Synthesismentioning

confidence: 72%

Methylation-dependent translation of viral messenger RNAs in vitro.

Both¹,

Banerjee²,

Shatkin³

1975

Proc. Natl. Acad. Sci. U.S.A.

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263

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Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wheat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation, S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypeptide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.Many animal viruses contain an RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) that transcribes the viral genome and produces RNA that functions as messenger in infected cells and in cell-free systems (1). Recently, it was reported that purified cytoplasmic polyhedrosis virus (2), reovirus (3), vaccinia virus (4, 5), and vesicular stomatitis virus (VSV) (6) also have an RNA methylase activity. Viral RNA synthesized in vitro in the presence of the methyl group donor S-adenosylmethionine (SAdoMet) is specifically methylated at the 5' end, resulting in molecules with 5'-terminal structures of the type m'G(5')ppp(5')Gm... and m7G(5')ppp (5' Synthesis and Purification of Viral mRNAs. The synthesis of reovirus mRNA by purified virus and its isolation by filtration through Sephadex G-50 and sedimentation in glycerol density gradients have been described (17). When methylated mRNA was required, 10 /uM SAdoIet was included in the transcription reaction mixture. VSV mRNA was synthesized as previously described (16) and purified by passage through Sephadex G-100 (6). VSV 12-18S mRNA was isolated by ethanol precipitation after sedimentation in a glycerol gradient (18). Methylated VSV mRNA was synthesized in the presence of 6.8 ,M SAdoMet (6).Translation of Viral mRNAs. Preparation of the wheat germ extract and the conditions for protein synthesis have been described (17,18). Reovirus and VSV mRNAs were used at concentrations of 100 and 40 Aug/ml, respectively, in a total incubation volume of 12.5,u. Protein synthesis in the presence of L-[5S]methionine (specific activity 200-400 Ci/mmol) was assayed by heating a 1 MuI aliquot of incubation mixture in 2 ml of 10% trichloroacetic acid containing 1% casamino acids (Difco, Detroit, Mich.) at 950 for 15 min. The samples were chilled in ice, collected on Millipore filters, and washed with 5 ml of trichloroacetic acid/casamino acids and 5 ml of 1% acetic acid. The filters were dried and their radioactivity was measured in a toluene-based scintillant.Methylation of Viral mRNAs by Cell-Free Extracts. Reo...

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Section: Dependence Of Methylation On Initiation Of Protein Synthesismentioning

confidence: 72%

Methylation-dependent translation of viral messenger RNAs in vitro.

Both¹,

Banerjee²,

Shatkin³

1975

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

263

View full text Add to dashboard Cite

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“…The capability of an aminoacyl adenylate to serve as an adenylylation intermediate for 5'-diphosphorylated RNAs is intriguing due to the recent observation of a m7G(5')ppp(5')Np group at the 5'-end of a variety of messenger RNAs (24,25). The parallelism of the in vitro capping of 70S avian myeloblastosis virus RNA mentioned above to the 5'-terminal guanylylation reaction of high-molecular-weight viral RNA presents an interesting subject for future investigation.…”

Section: Discussionmentioning

confidence: 99%

Relationship of the first step in protein synthesis to ppGpp: formation of A(5')ppp(5')Gpp.

Rapaport¹,

Svihovec²,

Zamecnik³

1975

Proc. Natl. Acad. Sci. U.S.A.

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In the presence of purified Escherichia coli lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP-forming) EC 6.1.1.6], L-lysine, and ATP, addition of the nucleotide ppGpp results in formation of a unique product-A(5')ppp(5') Gpp. The same compound is also formed very rapidly in a cell-free protein-synthesizing system when ppGpp is added. The possible significance of this reaction in the rapid turnover of ppGpp and as a more general mechanism by which an AMP residue is activated and introduced onto a 5'-diphosphorylated species, including the 5'-end of an RNA, is further discussed.

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“…The bracketed fractions in each of the left panels were pooled and used to obtain the analyses in the corresponding right panels by digestion with Pi nuclease and alkaline phosphatase and high voltage paper electrophoresis (24). 24 Nucleic Acids Research (Fig. 7C) and was partially degraded by treatment of incubation mixtures with nucleotide pyrophosphatase (Fig.…”

Section: '3mentioning

confidence: 99%

“…After centrifugation, fractions were collected and aliquots were counted. The bracketed fractions in each of the left panels were pooled and used to obtain the analyses in the corresponding right panels by digestion with Pi nuclease and alkaline phosphatase and high voltage paper electrophoresis (24). 24 Nucleic Acids Research (Fig.…”

Section: '3mentioning

confidence: 99%

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

et al. 1979

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A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.

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Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-.

Cited by 314 publications

References 26 publications

Methylation-dependent translation of viral messenger RNAs in vitro.

Methylation-dependent translation of viral messenger RNAs in vitro.

Relationship of the first step in protein synthesis to ppGpp: formation of A(5')ppp(5')Gpp.

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

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