N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

中文翻译：

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人类线粒体中t6A修饰的分子基础。

N 6-苏糖氨甲酰腺苷（t6A）是通用的tRNA修饰，对翻译的准确性和保真度至关重要。在人线粒体中，YrdC合成了一个1-苏氨酰氨基甲酰基腺苷酸（TC-AMP）中间体，OSGEPL1将TC部分转移到五个tRNA，包括人线粒体tRNAThr（hmtRNAThr）。hmtRNA，YrdC和OSGEPL1的突变影响了有效的t6A修饰，已涉及多种人类疾病。然而，关于人类线粒体中t6A形成中的tRNA识别机制知之甚少。在本文中，我们通过研究hmtRNAThr反密码子环中各个碱基对t6A修饰的贡献，表明OSGEPL1是单体，并且在利用C34作为抗决定剂方面具有独特性。通过在hmtRNAIle或A28中引入G38A，大大提高了OSGEPL1的活性：包含hmtRNASer（AGY）反密码子茎的嵌合tRNA中的U42碱基对，表明针对特定修饰水平对特定hmtRNA的序列进行了微调。此外，使用纯化的OSGEPL1，我们鉴定了多个乙酰化位点，并且OSGEPL1活性很容易受体外和体内多种机制的乙酰化影响。集体，我们系统地阐明了hmtRNA的反密码子环中的核苷酸要求，并揭示了涉及tRNA序列优化和翻译后蛋白质修饰（确定t6A修饰水平）的机制。OSGEPL1的活性很容易在体外和体内通过多种机制被乙酰化影响。集体，我们系统地阐明了hmtRNA的反密码子环中的核苷酸要求，并揭示了涉及tRNA序列优化和翻译后蛋白质修饰（确定t6A修饰水平）的机制。OSGEPL1的活性很容易在体外和体内通过多种机制被乙酰化影响。集体，我们系统地阐明了hmtRNA的反密码子环中的核苷酸要求，并揭示了涉及tRNA序列优化和翻译后蛋白质修饰（确定t6A修饰水平）的机制。