Csepany T, Lin A, Baldick C J, Beemon K
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J Biol Chem. 1990 Nov 25;265(33):20117-22.
The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells.
鸡mRNA N6-腺苷甲基转移酶的序列特异性已在体内进行了研究。在劳斯肉瘤病毒（RSV）病毒粒子RNA上六个新的N6-甲基腺苷位点的定位证实了我们扩展的甲基化共有序列：RGACU，其中R通常为G（7/12）。我们还在-2位置（相对于+1处的m6A）观察到A（2/12）和U（3/12），但从未观察到C。在+3位置，U出现了10/12次；在弱甲基化序列中，A和C各出现了一次。不同位点的甲基化程度有所不同，最高可达约90%。为了测试这个共有序列的重要性，通过位点特异性诱变对其进行了改变，并在将突变的RSV DNA转染到鸡胚成纤维细胞后检测甲基化情况。我们发现，将-1处的G或+3处的U替换为任何其他残基都会抑制甲基化。然而，抑制RSV src基因中所有四个主要位点的甲基化并未显著改变三种病毒RNA种类的稳态水平或病毒感染性。使src蛋白激酶活性失活的其他突变体产生的病毒较少，并且在感染细胞中src mRNA相对较少。
