Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus.
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 monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta 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 beta 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'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-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'-0-methylnucleoside residues per average mRNA molecule.
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000032:analysis*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D008113:Liver Neoplasms; D008745:Methylation; D009374:Neoplasms, Experimental / Q000032:analysis; D011061:Poly A / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D014885:Waxes
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014885:Waxes; D011061:Poly A