title: Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells.
abstract: The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-(3)H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2'-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2'-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2'-O-methylnucleoside fraction produced four peaks coincident with the four 2'-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N(6)-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives; D006528:Carcinoma, Hepatocellular / Q000032:analysis*; D002458:Cell Fractionation; D002460:Cell Line; D002482:Cellulose; D002845:Chromatography; D003562:Cytidine / Q000031:analogs & derivatives; D006151:Guanosine / Q000031:analogs & derivatives; D008113:Liver Neoplasms; D008745:Methylation; D009374:Neoplasms, Experimental / Q000032:analysis; D009705:Nucleosides / Q000032:analysis*; D009841:Oligonucleotides; D011132:Polyribosomes / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D013942:Thymine Nucleotides
chemical_list: D009705:Nucleosides; D009841:Oligonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013942:Thymine Nucleotides; D006151:Guanosine; D003562:Cytidine; D002482:Cellulose; D000241:Adenosine