Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells

R. Desrosiers, K. Friderici, F. Rottman

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Abstract

The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L [methyl 3H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT) cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE cellulose (borate) chromatography, which separates 2' O methylnucleosides from normal and base methylated nucleosides, about 50% of the radioactivity was recovered in the 2' O methylnucleoside fraction and 50% in the base methylnucleoside fraction. High speed liquid chromatography (Aminex A-5) of the 2' O methylnucleoside fraction produced four peaks coincident with the four 2' O methylnucleoside standards. Analysis of the base methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N6 methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.

Original languageEnglish (US)
Pages (from-to)3971-3975
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume71
Issue number10
DOIs
StatePublished - 1974

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