Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA

Abstract

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2′-0-methylribonucleosides, but only two basemethylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyllabeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5′ terminus of the poly(A)+ RNA and could be removed by periodate oxidation and β elimination. Evidence for a 5′ to 5′ linkage of 7MeG to adjacent 2′-0-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5′ppp5′NMep- and 7MeG5′ppp5′NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5′ termini may be a characteristic feature of mRNAs that function in eucaryotic cells. © 1975.