Sequences containing methylated nucleotides at the 5′ termini of messenger RNAs: Possible implications for processing

Rottman, Fritz M.; Shatkin, Aaron J.; Perry, Robert P.

doi:10.1016/0092-8674(74)90131-7

Cited by 262 publications

(205 citation statements)

References 8 publications

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“…. + p [20,27] Alternatively, the uncapped derivatives might be artifactually generated by cleavage of pyrophosphate bonds during alkaline hydrolysis or alkaline phosphatase treatment. However, in this regard, we did not observe a significant difference in their proportion when the concentration of alkaline phosphatase and the duration of incubation were both reduced concomitantly by factors of four.…”

Section: Discussionmentioning

confidence: 99%

The methylated constituents of globin mRNA

Perry

Scherrer

1975

FEBS Letters

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Section: Discussionmentioning

confidence: 99%

The methylated constituents of globin mRNA

Perry

Scherrer

1975

FEBS Letters

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“…Also, like mRNAs, hnRNAs were found to have the 7-methyl guanine 5′-5′ phosphotriester "cap" structure at their 5′ ends, like mRNAs. Remarkably, the methyl groups in hnRNA cap structures appeared to be conserved as RNA was exported to the cytoplasm, even though, on average, hnRNA molecules in the nucleus are at least four times longer than mRNAs in the cytoplasm (7,8). However, it was not possible to prove rigorously from the flow of radiolabel in pulse-chase labeling experiments that hnRNAs are precursors to mRNAs.…”

mentioning

confidence: 98%

Discovery of RNA splicing and genes in pieces

Berk

2016

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

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The Problem: Short-Lived Heterogeneous Nuclear RNA During the 1960s and 1970s, there was a major conundrum in eukaryotic molecular biology regarding the synthesis of mRNA in animal cells. Pulse and pulsechase radio-labeling experiments with tritiated uridine had shown that a large fraction of nuclear RNA was degraded very rapidly after synthesis, whereas only a small fraction of the initially synthesized RNA was exported from the nucleus to the cytoplasm where it functions as far more stable mRNAs (1, 2). This shortlived nuclear RNA ranged in lengths up to several tens of kilobases, much longer than most cytoplasmic mRNAs, and was referred to as heterogeneous nuclear RNA (hnRNA) (3, 4). Pulse-chase labeling yielded results consistent with hnRNA functioning as a precursor of mRNA. hnRNA is rapidly labeled, whereas label appeared in mRNAs more slowly. Also, as for mRNA, a large fraction of hnRNA was shown to be polyadenylated at its 3′ end like mRNA, consistent with the model that RNA sequences near the 3′ end of hnRNAs are retained in shorter mRNAs exported to the cytoplasm (5, 6). Also, like mRNAs, hnRNAs were found to have the 7-methyl guanine 5′-5′ phosphotriester "cap" structure at their 5′ ends, like mRNAs. Remarkably, the methyl groups in hnRNA cap structures appeared to be conserved as RNA was exported to the cytoplasm, even though, on average, hnRNA molecules in the nucleus are at least four times longer than mRNAs in the cytoplasm (7, 8). However, it was not possible to prove rigorously from the flow of radiolabel in pulse-chase labeling experiments that hnRNAs are precursors to mRNAs. There were two principal reasons for this. First, the pool of ribonucleoside triphosphates in mammalian cells is so large that it takes more than 30 min to saturate the pools when labeled nucleosides are added to the media and equally long to dilute the intracellular pool of labeled nucleoside triphosphates by addition of a large excess of unlabeled nucleoside to the culture medium during the chase period. Consequently, a clean chase could not be achieved, and newly synthesized hnRNAs continued to be labeled as labeled mRNAs first appeared in the cytoplasm. Also, the small fraction of pulse-labeled nuclear RNA converted to more stable mRNAs, only 5-10%, added to the difficulty of proving a precursor product relationship from pulse-chase labeling experiments.The synthesis and processing of ribosomal RNAs was much easier to analyze than processing of hnRNAs because about 50% of all nuclear RNA synthesis in growing mammalian cells in culture is synthesis of the single ∼13.7-kb pre-rRNA precursor. Moreover, this full-length pre-rRNA precursor accumulates to significant levels before it is processed into stable mature rRNAs of 18S (1,870 bases), 5.8S (156 bases), and 28S (5,034 bases), so that the fraction of pre-RNA processed into stable cytoplasmic rRNAs is ∼50%, much more than the 5-10% of pulse-labeled hnRNA processed into mRNAs. This fortuitous situation for the study of prerRNA by pulse-chase labeling made it possible to ...

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“…The m7Gp is derived from GTP, apparently by a "capping process" (14). If cell-free extracts can convert viral mRNAs with unblocked, unmethylated 5' termini to chains containing 5'-terminal m7Gp, it may explain, at least in part, the GTP requirement for initiation of polypeptide chains (21).…”

Section: Dependence Of Methylation On Initiation Of Protein Synthesismentioning

confidence: 99%

Methylation-dependent translation of viral messenger RNAs in vitro.

Both¹,

Banerjee²,

Shatkin³

1975

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

262

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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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Sequences containing methylated nucleotides at the 5′ termini of messenger RNAs: Possible implications for processing

Cited by 262 publications

References 8 publications

The methylated constituents of globin mRNA

The methylated constituents of globin mRNA

Discovery of RNA splicing and genes in pieces

Methylation-dependent translation of viral messenger RNAs in vitro.

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