Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus

作者：Ronald C. Desrosiers, Karen H. Friderici, FRITZ M. ROTTMAN · 发表于：Biochemistry · 年份：1975 · DOI：10.1021/bi00691a004 · 被引用次数：194 · 研究领域：RNA modifications and cancer、Cancer-related gene regulation、RNA Research and Splicing

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-methylnucl...