Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
J Mol Biol. 2010 Feb 5;395(5):1038-48. doi: 10.1016/j.jmb.2009.12.002. Epub 2009 Dec 11.
Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)(5)s(2)U(34) in mitochondrial tRNA(Lys), tRNA(Glu), and tRNA(Gln). In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm(5) group (cmnm(5)U(34)), while Mto2p is responsible for the 2-thiouridylation (s(2)U(34)) of these tRNAs. Previous studies showed that partial modifications at U(34) in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA(Lys), tRNA(Glu), and tRNA(Gln) in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln), caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm(5)U(34) by the mto1 or mss1 mutation. The complete loss of modifications at U(34) of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln) in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.
酿酒酵母 MTO2、MTO1 和 MSS1 基因编码高度保守的 tRNA 修饰酶，用于线粒体 tRNA（Lys）、tRNA（Glu）和 tRNA（Gln）中羧甲基氨基甲酰甲基（cmnm）（5）s（2）U（34）的生物合成。事实上，Mto1p 和 Mss1p 参与 cmnm（5）基团（cmnm（5）U（34））的生物合成，而 Mto2p 负责这些 tRNA 的 2-硫代尿嘧啶化（s（2）U（34））。先前的研究表明，线粒体 tRNA 中 U（34）的部分修饰使 mto1、mto2 和 mss1 菌株能够呼吸。在本报告中，我们通过使用 mto2、mto1 和 mss1 单突变体、双突变体和三突变体研究了 MTO2、MTO1 和 MSS1 基因之间的功能相互作用。令人惊讶的是，在含有甘油的培养基中，MTO2 的缺失与 MSS1 或 MTO1 的缺失具有合成致死性，但在含有葡萄糖的培养基中则没有。有趣的是，在 mto2/mss1、mto2/mto1 和 mto2/mto1/mss1 菌株中，没有检测到包括 tRNA（Lys）、tRNA（Glu）和 tRNA（Gln）在内的九种 tRNA。此外，mto2/mss1、mto2/mto1 和 mto2/mto1/mss1 突变体表现出 COX1 和 CYTB mRNA 以及 15S 和 21S rRNA 的极低水平，以及线粒体蛋白质合成的完全丧失。这种合成增强组合可能是由于 mto2 突变消除了 2-硫代尿嘧啶化，而 mto1 或 mss1 突变导致 tRNA（Lys）、tRNA（Glu）和 tRNA（Gln）中 U（34）的修饰完全缺失所致。tRNA 中 U（34）修饰的完全缺失改变了线粒体 RNA 代谢，导致线粒体 tRNA、mRNA 和 rRNA 的降解。结果，线粒体 RNA 代谢的失败导致线粒体翻译的完全丧失。因此，线粒体蛋白质合成的缺陷导致其线粒体基因组的不稳定，从而产生呼吸缺陷表型。因此，我们的研究结果表明，tRNA（Lys）、tRNA（Glu）和 tRNA（Gln）中 U（34）的修饰在维持线粒体基因组、线粒体 RNA 稳定性、翻译和呼吸功能方面发挥着关键作用。
