Fidelity of translation initiation is required for coordinated respiratory complex assembly

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs. To date it is not clear how mitoribosomes recognize and initiate translation in the absence of untranslated regions in the mitochondrial mRNAs. Translation initiation in mitochondria shares similarities with prokaryotic systems, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors, MTIF2 that closes the decoding centre and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3 whose role is not clear. We knocked out Mtif3 in mice and show that this protein is essential for embryo development and heart- and skeletal muscle-specific loss of MTIF3 causes premature death. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3 that results in loss of specific respiratory complexes. Therefore, we show that coordinated assembly of OXPHOS complexes requires stoichiometric levels of nuclear and mitochondrially-encoded protein subunits in vivo. Our ribosome profiling and transcriptomic analyses show that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs, but not dissociation of the ribosome subunits. Total RNA was isolated from heart tissue from 25 week old control (Mtif3loxP/loxP) and Mtif3 knockout mice (Mtif3loxP/loxP, +/Ckmm), and TruSeq libraries produced, sequenced and analysed for differential expression. Sucrose gradient fractions of mitochondrial RNA corresponding to small and large subunit, as well as assembled ribosomes, was isolated from heart tissue from 25 week old control (Mtif3loxP/loxP) and Mtif3 knockout mice (Mtif3loxP/loxP, +/Ckmm), and ribosome profiling libraries produced and sequenced.哺乳动物线粒体核糖体是一类独特的分子机器，负责翻译11种无引导序列的mRNA（messenger RNA）。迄今为止，学界尚未明确线粒体核糖体如何在线粒体mRNA缺乏非翻译区的情况下识别并启动翻译过程。线粒体翻译起始过程与原核系统具有相似性，例如可形成fMet-tRNAMet（甲酰甲硫氨酸转运RNA）、mRNA与28S亚基组成的三元复合物，但在起始因子的需求层面存在差异。线粒体仅拥有两种起始因子：MTIF2（线粒体翻译起始因子2）可封闭解码中心并稳定fMet-tRNAMet与无引导序列mRNA的结合，而MTIF3（线粒体翻译起始因子3）的功能目前尚不明确。我们在小鼠体内敲除Mtif3基因，结果显示该蛋白对于胚胎发育不可或缺；心脏与骨骼肌特异性缺失MTIF3则会导致小鼠过早死亡。我们发现，缺失MTIF3的小鼠线粒体蛋白质合成水平升高但调控失衡，进而造成特定呼吸链复合物的缺失。因此，本研究证实，氧化磷酸化（OXPHOS）复合物的协同组装在活体中需要核编码与线粒体编码的蛋白质亚基维持化学计量比水平。我们的核糖体谱分析与转录组分析结果表明，MTIF3对于线粒体mRNA翻译起始的识别与调控必不可少，但并不参与核糖体亚基的解离过程。我们从25周龄对照小鼠（Mtif3loxP/loxP）以及Mtif3敲除小鼠（Mtif3loxP/loxP, +/Ckmm）的心脏组织中提取总RNA，构建TruSeq测序文库并完成测序，随后进行差异表达分析；同时从上述两组小鼠的心脏组织中分离得到对应线粒体核糖体小亚基、大亚基以及组装完成核糖体的蔗糖梯度分级组分，构建核糖体谱测序文库并完成测序。