Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1985 Sep; 5(9): 2298–2306.
PMCID: PMC366956

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.


N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aloni Y, Dhar R, Khoury G. Methylation of nuclear simian virus 40 RNAs. J Virol. 1979 Oct;32(1):52–60. [PMC free article] [PubMed]
  • Banerjee AK. 5'-terminal cap structure in eucaryotic messenger ribonucleic acids. Microbiol Rev. 1980 Jun;44(2):175–205. [PMC free article] [PubMed]
  • Beemon K, Keith J. Localization of N6-methyladenosine in the Rous sarcoma virus genome. J Mol Biol. 1977 Jun 15;113(1):165–179. [PubMed]
  • Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. [PubMed]
  • Camper SA, Albers RJ, Coward JK, Rottman FM. Effect of undermethylation on mRNA cytoplasmic appearance and half-life. Mol Cell Biol. 1984 Mar;4(3):538–543. [PMC free article] [PubMed]
  • Canaani D, Kahana C, Lavi S, Groner Y. Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA. Nucleic Acids Res. 1979 Jun 25;6(8):2879–2899. [PMC free article] [PubMed]
  • Casey J, Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. [PMC free article] [PubMed]
  • Chen-Kiang S, Nevins JR, Darnell JE., Jr N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. J Mol Biol. 1979 Dec 15;135(3):733–752. [PubMed]
  • Darnell JE., Jr Transcription units for mRNA production in eukaryotic cells and their DNA viruses. Prog Nucleic Acid Res Mol Biol. 1979;22:327–353. [PubMed]
  • DeLorbe WJ, Luciw PA, Goodman HM, Varmus HE, Bishop JM. Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J Virol. 1980 Oct;36(1):50–61. [PMC free article] [PubMed]
  • Dimock K, Stoltzfus CM. Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits. Biochemistry. 1977 Feb 8;16(3):471–478. [PubMed]
  • Finkel D, Groner Y. Methylations of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs. Virology. 1983 Dec;131(2):409–425. [PubMed]
  • Furuichi Y, Shatkin AJ, Stavnezer E, Bishop JM. Blocked, methylated 5'-terminal sequence in avian sarcoma virus RNA. Nature. 1975 Oct 16;257(5527):618–620. [PubMed]
  • Gillespie D, Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. [PubMed]
  • Hayward WS, Neel BG. Retroviral gene expression. Curr Top Microbiol Immunol. 1981;91:217–276. [PubMed]
  • Horowitz S, Horowitz A, Nilsen TW, Munns TW, Rottman FM. Mapping of N6-methyladenosine residues in bovine prolactin mRNA. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5667–5671. [PMC free article] [PubMed]
  • Katz RA, Omer CA, Weis JH, Mitsialis SA, Faras AJ, Guntaka RV. Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions. J Virol. 1982 Apr;42(1):346–351. [PMC free article] [PubMed]
  • Keller EB, Noon WA. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417–7420. [PMC free article] [PubMed]
  • Laskey RA. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 1980;65(1):363–371. [PubMed]
  • Messing J, Gronenborn B, Müller-Hill B, Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. [PMC free article] [PubMed]
  • Messing J, Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. [PubMed]
  • Moss B, Gershowitz A, Weber LA, Baglioni C. Histone mRNAs contain blocked and methylated 5' terminal sequences but lack methylated nucleosides at internal positions. Cell. 1977 Jan;10(1):113–120. [PubMed]
  • Norrander J, Kempe T, Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. [PubMed]
  • Padgett RA, Konarska MM, Grabowski PJ, Hardy SF, Sharp PA. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. [PubMed]
  • Perry RP, Scherrer K. The methylated constituents of globin mRNA. FEBS Lett. 1975 Sep 1;57(1):73–78. [PubMed]
  • Revel M, Groner Y. Post-transcriptional and translational controls of gene expression in eukaryotes. Annu Rev Biochem. 1978;47:1079–1126. [PubMed]
  • Ruskin B, Krainer AR, Maniatis T, Green MR. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. [PubMed]
  • Sanger F, Coulson AR, Barrell BG, Smith AJ, Roe BA. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. [PubMed]
  • Schibler U, Kelley DE, Perry RP. Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. J Mol Biol. 1977 Oct 5;115(4):695–714. [PubMed]
  • Schwartz DE, Tizard R, Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. [PubMed]
  • Sommer S, Salditt-Georgieff M, Bachenheimer S, Darnell JE, Furuichi Y, Morgan M, Shatkin AJ. The methylation of adenovirus-specific nuclear and cytoplasmic RNA. Nucleic Acids Res. 1976 Mar;3(3):749–765. [PMC free article] [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Stoltzfus CM, Dane RW. Accumulation of spliced avian retrovirus mRNA is inhibited in S-adenosylmethionine-depleted chicken embryo fibroblasts. J Virol. 1982 Jun;42(3):918–931. [PMC free article] [PubMed]
  • Stoltzfus CM, Dimock K. Evidence of methylation of B77 avian sarcoma virus genome RNA subunits. J Virol. 1976 May;18(2):586–595. [PMC free article] [PubMed]
  • Wallace JC, Edmonds M. Polyadenylylated nuclear RNA contains branches. Proc Natl Acad Sci U S A. 1983 Feb;80(4):950–954. [PMC free article] [PubMed]
  • Wei CM, Moss B. Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid. Biochemistry. 1977 Apr 19;16(8):1672–1676. [PubMed]
  • Zeitlin S, Efstratiadis A. In vivo splicing products of the rabbit beta-globin pre-mRNA. Cell. 1984 Dec;39(3 Pt 2):589–602. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...