Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA
Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA
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DOI：
10.1017/s1355838202024019
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发表时间：
2002-10-01
期刊：
影响因子：
4.5
通讯作者：
Phizicky, EM
中科院分区：
文献类型：
--
作者：
Alexandrov, A;Martzen, MR;Phizicky, EM
tRNA的7 - 甲基鸟苷（m(7)G）修饰在真核生物和细菌中广泛存在，几乎总是位于第46位，是少数能给碱基赋予正电荷的修饰之一。对纯化的GST - ORF融合蛋白的酿酒酵母基因组文库进行筛选，发现了两种先前未被鉴定的蛋白质，它们与前体tRNA（Phe）上的m(7)G甲基转移酶活性共同纯化。开放阅读框YDL201w编码Trm8，这是一种在原核生物和真核生物中高度保守的蛋白质，含有一个S - 腺苷甲硫氨酸结合域。开放阅读框YDR165w编码Trm82，这是一种保守性较低的蛋白质，含有假定的WD40重复序列，这些重复序列通常与大分子相互作用有关。这两种蛋白质与催化mRNA 5'帽m(7)G修饰的酵母Abd1都没有显著的序列相似性，除了Trm8和Abd1共有的甲基转移酶基序。几条证据表明，Trm8和Trm82蛋白质对于tRNA的m(7)G - 甲基转移酶活性都是必需的：来自缺失任一基因的菌株的提取物都检测不到m(7)G甲基转移酶活性，缺失任一基因的菌株的RNA中m(7)G含量大幅降低，并且需要两种蛋白质共同表达才能过量产生活性。苯胺裂解图谱显示，Trm8/Trm82蛋白质在G46（体内被修饰的位点）修饰前体tRNA（Phe）。Trm8和Trm82蛋白质形成一个复合物，因为Trm8蛋白质的亲和纯化会导致Trm82蛋白质以大致等摩尔的量共同纯化。这种功能性的双蛋白家族似乎在真核生物中得以保留，因为相应的两个人类蛋白质METTL1和WDR4的表达对于m(7)G - 甲基转移酶活性是必需的。
7-methylguanosine (m(7)G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m(7)G methyltransferase activity on pre-tRNA(Phe). ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m(7)G modification of the 5' cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m(7)G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m(7)G methyltransferase activity, RNA from strains lacking either gene have much reduced m(7)G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNA(Phe) at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m(7)G-methyltransferase activity.