title: Effect of undermethylation on mRNA cytoplasmic appearance and half-life.
abstract: S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006207:Half-Life; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006710:Homocysteine / Q000031:analogs & derivatives*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D012333:RNA, Messenger; D012334:RNA, Neoplasm; D006710:Homocysteine; D011061:Poly A; C012086:S-tubercidinylhomocysteine; D012313:RNA; D012435:S-Adenosylhomocysteine