EFFECT OF UNDERMETHYLATION ON MESSENGER-RNA CYTOPLASMIC APPEARANCE AND HALF-LIFE
EFFECT OF UNDERMETHYLATION ON MESSENGER-RNA CYTOPLASMIC APPEARANCE AND HALF-LIFE
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DOI：
10.1128/mcb.4.3.538
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发表时间：
1984-01-01
影响因子：
5.3
通讯作者：
ROTTMAN, FM
中科院分区：
文献类型：
--
作者：
CAMPER, SA;ALBERS, RJ;ROTTMAN, FM
S-同型半胱氨酸是S-腺苷同型半胱氨酸的结构类似物，是S-腺苷蛋氨酸依赖的甲基转移酶反应的有效抑制剂。观察STH对HeLa细胞mRNA代谢的影响。双重标记研究表明，STH以剂量依赖的方式显著抑制HeLa mRNA的甲基化。用高压液相色谱分析修饰的核苷和5‘-末端帽结构表明，腺苷的内部N6-甲基化在50µM和500µM STH分别减少了65%和83%。帽子结构中腺苷的N6-甲基化在两种浓度的STH中都有类似的降低。在Sth的较高水平上检测到大量的2‘’-O-甲基化核苷(m7GpppN，cap0)的帽子结构松弛。为了测试甲基化影响mRNA稳定性的可能性，在脉冲追逐实验中测量了细胞质mRNA的半衰期。与对照组相比，STH处理后产生的甲基化不足的mRNA的半衰期没有变化。为了确定mRNA甲基化是否与核加工或转运有关，比较了STH处理的HeLa细胞和未处理的HeLa细胞中多聚腺苷RNA出现在细胞质中的时间。STH导致多腺化RNA出现的时间明显滞后，表明mRNA甲基化可能是有效处理或运输所必需的。
S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocystine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. The effects of STH on HeLa cell mRNA metabolism were investigated. 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 .mu.M STH and by 83% at 500 .mu.M STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structure slacking 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 treament, 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.