The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs
The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs
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DOI：
10.1017/s1355838299981475
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发表时间：
1999-01-01
期刊：
影响因子：
4.5
通讯作者：
Bachellerie, JP
中科院分区：
文献类型：
--
作者：
Cavaillé, J;Chetouani, F;Bachellerie, JP
三种已知的RNA2‘-O-核糖甲基酶的蛋白质序列被用作探针，通过重复搜索基因组数据库来检测可能的同源物。我们已经确定了45个新的正向开放阅读框架(ORF)，主要存在于原核基因组中。还检测到5个完整的真核ORF，其中在酿酒酵母基因组中只有一个ORF(YDL112w)。在YDL112w基因缺失后，我们观察到了tRNA核糖甲基化的一个特殊缺陷，在所有自然含有这种修饰的tRNA中，Gm18完全消失，而其他tRNA核糖甲基化和rRNA核糖甲基化的复杂模式没有受到影响。用允许表达YDL112wp的质粒转化破坏的菌株，抑制了tRNA G18甲基化缺陷。在天然含有这种甲基化的酵母tRNA(Ser)的体外转录本上形成Gm18，这是由野生型酵母菌株的无细胞提取物有效地催化的，而不是用破坏菌株的提取物发生的。因此，YDL112w ORF编码的蛋白质称为Trm3(tRNA甲基化)，很可能是tRNA(Gm18)核糖甲基酶。在体外实验中，其活性强烈依赖于tRNA结构。Trm3p是在真核生物中发现的第一个假定的tRNA核糖甲基化酶，它比它的大肠杆菌功能同源物spou(1,436个氨基酸vs.229个氨基酸)或任何已知或推测的原核RNA核糖甲基转移酶都要大得多。在人类(Trp-185蛋白)、秀丽线虫和拟南芥中发现的同源物也显示出一个很长的N末端延伸，与数据库中的任何蛋白质序列都不相关。
The protein sequences of three known RNA 2'-O-ribose methylases were used as probes for detecting putative homologs through iterative searches of genomic databases. We have identified 45 new positive Open Reading Frames (ORFs), mostly in prokaryotic genomes. Five complete eukaryotic ORFs were also detected, among which was a single ORF (YDL112w) in the yeast Saccharomyces cerevisiae genome. After genetic depletion of YDL112w, we observed a specific defect in tRNA ribose methylation, with the complete disappearance of Gm18 in all tRNAs that naturally contain this modification, whereas other tRNA ribose methylations and the complex pattern of rRNA ribose methylations were not affected. The tRNA G18 methylation defect was suppressed by transformation of the disrupted strain with a plasmid allowing expression of YDL112wp. The formation of Gm18 on an in vitro transcript of a yeast tRNA(Ser) naturally containing this methylation, which was efficiently catalyzed by cell-free extracts from the wild-type yeast strain, did not occur with extracts from the disrupted strain. The protein encoded by the YDL112w ORF, termed Trm3 (tRNA methylation), is therefore likely to be the tRNA (Gm18) ribose methylase. In in vitro assays, its activity is strongly dependent on tRNA architecture. Trm3p, the first putative tRNA ribose methylase identified in an eukaryotic organism, is considerably larger than its Escherichia coli functional homolog spoU (1,436 amino acids vs. 229 amino acids), or any known or putative prokaryotic RNA ribose methyltransferase. Homologs found in human (TRP-185 protein), Caenorhabditis elegans and Arabidopsis thaliana also exhibit a very long N-terminal extension not related to any protein sequence in databases.