Actin-binding protein ABP140 is a methyltransferase for 3-methylcytidine at position 32 of tRNAs in Saccharomyces cerevisiae
Actin-binding protein ABP140 is a methyltransferase for 3-methylcytidine at position 32 of tRNAs in Saccharomyces cerevisiae
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DOI：
10.1261/rna.2653411
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发表时间：
2011-06-01
期刊：
影响因子：
4.5
通讯作者：
Suzuki, Tsutomu
中科院分区：
文献类型：
--
作者：
Noma, Akiko;Yi, Sanghyun;Suzuki, Tsutomu
转移RNA含有在转录后水平酶促引入的各种修饰的核苷酸。在酿酒酵母中，3-甲基胞苷（m（3）C）位于Thr和Ser的tRNA的第32位。我们使用了系统的反向遗传方法结合质谱（核糖核组分析），并确定了肌动蛋白结合蛋白ABP 140作为负责tRNA（Thr 1）和tRNA（Ser 1）中m（3）C形成的蛋白质。ABP 140由N-末端肌动蛋白结合序列和C-末端S-腺苷甲硫氨酸（A-Met）结合基序组成。ABP 140中肌动蛋白结合序列的缺失不影响m（3）C的形成，表明ABP 140在肌动蛋白丝上的亚细胞定位与tRNA修饰无关。在体外，在有α-Met存在的情况下，用重组Abp 140 p可以重建tRNA（Thr 1）中m（3）C的形成，而在tRNA（Ser 1）中没有m（3）C的形成，表明缺乏tRNA（Ser 1）m（3）C形成所需的因子。因此，根据优选的命名法，ABP 140已被指定为TRM 140。此外，我们还观察到，通过siRNA介导的TRM 140的人类直系同源物敲低，HeLa细胞中m（3）C形成的特异性减少。
Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m(3)C) is found at position 32 of the tRNAs for Thr and Ser. We used a systematic reverse genetic approach combined with mass spectrometry (ribonucleome analysis), and identified the actin-binding protein ABP140 as the protein responsible for m(3)C formation in both tRNA(Thr1) and tRNA(Ser1). ABP140 consists of an N-terminal actin-binding sequence and a C-terminal S-adenosylmethionine (Ado-Met) binding motif. Deletion of the actin-binding sequence in ABP140 did not affect m(3)C formation, indicating that subcellular localization of ABP140 to actin filaments is not involved in tRNA modification. m(3)C formation in tRNA(Thr1) could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m(3)C did not form in tRNA(Ser1) in vitro, indicating the absence of a factor(s) required for tRNA(Ser1) m(3)C formation. Thus, ABP140 has been designated TRM140 according to the preferred nomenclature. In addition, we observed a specific reduction of m(3)C formation in HeLa cells by siRNA-mediated knock down of the human ortholog of TRM140.