Lee Muhoon, Wakigawa Taisei, Jia Qimin, Liu Chang, Huang Ruiyuan, Huang Shuai, Nagao Asuteka, Suzuki Tsutomu, Tomita Kozo, Iwasaki Shintaro, Takeuchi-Tomita Nono
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf021.
The mammalian mitochondrial protein synthesis system produces 13 essential subunits of oxidative phosphorylation (OXPHOS) complexes. Translation initiation in mammalian mitochondria is characterized by the use of leaderless messenger RNAs (mRNAs) and non-AUG start codons, where the proofreading function of IF-3mt still remains elusive. Here, we developed a reconstituted mammalian mitochondrial translation system using in vitro transcribed and native mitochondrial transfer RNAs (tRNAs) to investigate IF-3mt's proofreading function. Similar to bacterial IF-3, IF-3mt permits an initiator tRNA to participate in initiation by discriminating the three G-C pairs in its anticodon stem, and by the cognate interactions of its anticodon with the AUG start codon. As a result, IF-3mt promotes the accurate initiation of leaderless mRNAs. Nevertheless, IF-3mt can also facilitate initiation from the non-AUG(AUA) start codon through its unique N- and C-terminal extensions, in concert with the 5-methylcytidine (m5C) or 5-formylcytidine (f5C) modification at the anticodon wobble position of mt-tRNAMet. This is partly because the IF-3mt-specific N- and C-terminal extensions and the KKGK-motif favor leaderless mRNA initiation and relax non-AUG start codon discrimination. Analyses of IF-3mt-depleted human cells revealed that IF-3mt indeed participates in translating the open reading frames (ORFs) of leaderless mRNAs, as well as the internal ORFs of dicistronic mRNAs.
哺乳动物线粒体蛋白质合成系统产生氧化磷酸化（OXPHOS）复合物的13个必需亚基。哺乳动物线粒体中的翻译起始以使用无帽信使核糖核酸（mRNA）和非AUG起始密码子为特征，其中IF-3mt的校对功能仍然难以捉摸。在这里，我们使用体外转录的天然线粒体转移核糖核酸（tRNA）开发了一种重组哺乳动物线粒体翻译系统，以研究IF-3mt的校对功能。与细菌IF-3类似，IF-3mt通过识别起始tRNA反密码子茎中的三个G-C对，以及其反密码子与AUG起始密码子的同源相互作用，允许起始tRNA参与起始。结果，IF-3mt促进了无帽mRNA的准确起始。然而，IF-3mt还可以通过其独特的N端和C端延伸，与mt-tRNAMet反密码子摆动位置的5-甲基胞嘧啶（m5C）或5-甲酰基胞嘧啶（f5C）修饰协同作用，促进从非AUG（AUA）起始密码子起始。部分原因是IF-3mt特异性的N端和C端延伸以及KKGK基序有利于无帽mRNA起始，并放松对非AUG起始密码子的识别。对缺乏IF-3mt的人类细胞的分析表明，IF-3mt确实参与翻译无帽mRNA的开放阅读框（ORF）以及双顺反子mRNA的内部ORF。
