KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the mononucleotide (53%) is due entirely to N6-methyIadenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2′,-O-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by β elimination released only labeled 7-methylguanine consistent with a blocked 5′ terminus containing an unusual 5′-5′ bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by β elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2′-O-methylnucleotides linked by a 5′-5′ bond to a terminal 7-mfethylguanosine and the 2′-O-methylation can occur with any of the four nucleotides. The 5′ terminus may be represented by m7G5′ ppp5′ (Nmp)lor2Np, a general structure proposed earlier as a possible 5′ terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2′-O- methylnucleoside residues per average mRNA molecule.
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