线粒体RNAm^(3)C修饰酶METTL8的活力依赖于亚型特异的N-末端延伸结构且METTL8能修饰多种非天然底物tRNA  

Mitochondrial RNA m3C methyltransferase METTL8 relies on an isoform-specific N-terminal extension and modifies multiple heterogenous tRNAs

作　　者：黄梦涵 王金涛 张剑辉 毛雪玲 彭桂鑫 林秀颖 吕岱竹 袁晨 林桓 王恩多 周小龙 Meng-Han Huang;Jin-Tao Wang;Jian-Hui Zhang;Xue-Ling Mao;Gui-Xin Peng;Xiuying Lin;Daizhu Lv;Chen Yuan;Huan Lin;En-Duo Wang;Xiao-Long Zhou(Key Laboratory of RNA Science and Engineering,State Key Laboratory of Molecular Biology,CAS Center for Excellence in Molecular Cell Science,Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,University of Chinese Academy of Sciences,Shanghai 200031,China;Key Laboratory of Systems Health Science of Zhejiang Province,School of Life Science,Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences,Hangzhou 310024,China;School of Life Science and Technology,ShanghaiTech University,Shanghai 201210,China;State Key Laboratory of Marine Resource Utilization in South China Sea,Hainan University,Haikou 570228,China;School of Life Sciences,Hainan University,Haikou 570228,China;Analysis and Testing Center,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China;School of Pharmacy,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]Key Laboratory of RNA Science and Engineering,State Key Laboratory of Molecular Biology,CAS Center for Excellence in Molecular Cell Science,Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,University of Chinese Academy of Sciences,Shanghai 200031,China [2]Key Laboratory of Systems Health Science of Zhejiang Province,School of Life Science,Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences,Hangzhou 310024,China [3]School of Life Science and Technology,ShanghaiTech University,Shanghai 201210,China [4]State Key Laboratory of Marine Resource Utilization in South China Sea,Hainan University,Haikou 570228,China [5]School of Life Sciences,Hainan University,Haikou 570228,China [6]Analysis and Testing Center,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China [7]School of Pharmacy,China Pharmaceutical University,Nanjing 211198,China

出　　处：《Science Bulletin》2023年第18期2094-2105,M0004,共13页科学通报（英文版）

基　　金：supported by the National Key Research and Development Program of China(2021YFA1300800 and 2021YFC2700903);the National Natural Science Foundation of China(32271300,91940302,31900436,and 81870896);the Committee of Science and Technology in Shanghai(22ZR1481300 and 22JC1400503);the Chinese Academy of Sciences(CAS)Project for Young Scientists in Basic Research(YSBR-075).

摘　　要：定位于线粒体的METTL8-Iso1和分布在核仁的METTL8-Iso4均为甲基转移酶基因METTL8通过mRNA选择性剪接产生的亚型,它们的区别仅为前者有N-末端延伸结构.METTL8-Iso1的功能是催化产生线粒体tRNATh和tRNASe(UCN)第32位3-甲基胞苷(mC32)修饰.METTL8-Iso4是否也具有tRNAmC32修饰活性以及N-末端延伸在线粒体tRNAmC32修饰中的作用尚不清楚。我们发现,N-末端延伸上有几个保守的关键氨基酸残基,而METTL8-Iso4由于缺少N-未端延伸而不具备tRNAmC32修饰活力.体外实验和体内实验的结果表明,甲基转移酶METTL2A和Trm140上对应的这些关键位点也是各自对底物tRNAmC32修饰活性所必需的。在跨细胞区室与跨物种的酶对tRNA体外催化的交又实验中,我们意外地发现METTL8-Iso1也能催化几种人细胞质tRNA甚至大肠杆菌tRNA的mC32修饰。此外,mC32修饰并不影响tRNA的N-苏氨酰氨基甲酰腺苷(t'A)修饰和氨基酰化活力.除了METTL8与人线粒体丝氨酰-tRNA合成酶(SARS2)的相互作用外,我们还进一步发现了人线粒体苏氨酰-tRNA合成酶(TARS2)与METTL8-IsoI之间的相互作用。体外实验中,METTL8-Iso1显著促进了SARS2和TARS2的氨酰化活力,表明线粒体IRNA的修饰和氨基酰化之间存在功能联系:总之,该结果加深了对线粒体mC32修饰生物发生机制的理解,并提供了一种制备仅含mC32修饰的人细胞质或细菌tRNA的方法,有助于未来研究mC32修饰对tRNA结构和功能的影响。Methyltransferase-like 8(METTL8)encodes a mitochondria-localized METTL8-Iso1 and a nucleolusdistributed METTL8-Iso4 isoform,which differ only in their N-terminal extension(N-extension),by mRNA alternative splicing.METTL8-Iso1 generates 3-methylcytidine at position 32(m3C32)of mitochondrial tRNAThr and tRNASer(UCN).Whether METTL8-Iso4 is an active m3C32 methyltransferase and the role of the N-extension in mitochondrial tRNA m3C32 formation remain unclear.Here,we revealed that METTL8-Iso4 was inactive in m3C32 generation due to the lack of N-extension,which contains several absolutely conserved modification-critical residues;the counterparts were likewise essential in cytoplasmic m^(3)C32 biogenesis by methyltransferase-like 2A(METTL2A)or budding yeasts tRNA N3-methylcytidine methyltransferase(Trm140),in vitro and in vivo.Cross-compartment/species tRNA modification assays unexpectedly found that METTL8-Iso1 efficiently introduced m^(3)C32 to several cytoplasmic or even bacterial tRNAs in vitro.m3C32 did not influence tRNAThr N6-threonylcarbamoyladenosine(t6A)modification or aminoacylation.In addition to its interaction with mitochondrial seryl-tRNA synthetase(SARS2),we further discovered an interaction between mitochondrial threonyl-tRNA synthetase(TARS2)and METTL8-Iso1.METTL8-Iso1 substantially stimulated the aminoacylation activities of SARS2 and TARS2 in vitro,suggesting a functional connection between mitochondrial tRNA modification and charging.Altogether,our results deepen the mechanistic insights into mitochondrial m^(3)C32 biogenesis and provide a valuable route to prepare cytoplasmic/bacterial tRNAs with only a m^(3)C32 moiety,aiding in future efforts to investigate its effects on tRNA structure and function.

关 键 词：TRNA 3-methylcytidine METHYLTRANSFERASE AMINOACYLATION 

分 类 号：Q75[生物学—分子生物学]