The methylation state of poly A-containing-messenger RNA from cultured hamster cells

• Published in: Nucleic acids research 1975-10, Vol.2 (10), p.1653-1668
• Main Authors: Dubin, Donald T., Taylor, Robert H.
• Format: Article
• Language: English
• Subjects: Cell Line; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Methylation; Poly A - analysis; Ribonucleases; RNA, Messenger - analysis
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/2.10.1653

The poly A-containing mRNA of cultured hamster (BHK-21) cells has been examined with regard to methylation status. Steady state-labeled mRNA was obtained by incubating cells for 20–22h in the presence of [methyl-3H]-methionine and 32Pi. The degree of methylation of this RNA was 1.8 methyl groups per 1000 nucleotides, or 4–5 methyl groups on the average per molecule. The nature of the methylated residues was determined by paper chromatography and electrophoresis of acid and alkaline hydrolysates, by DEAE cellulose chromatography of alkaline hydrolysates and of T2 RNase digests, and by examining the effect of subjecting samples to “β-elimination.” Approx. half of the methyl groups occurred in standard (“internal”) linkage, 10% as m5Cp and 40% as m6Ap residues. The remainder occurred at least for the most part in “blocked” 5′-termini with the presumptive structure m7G(5′)ppp(Nm)p.., where Nm was Gm, m6Am, Um, or Cm.