5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA

The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.