Reinsch Julia L, Garcia David M
bioRxiv. 2025 May 10:2025.05.09.653160. doi: 10.1101/2025.05.09.653160.
Mutations in tRNA-modifying enzymes are associated with severe mitochondrial disease in humans. has been used to model the impact of these mutations due to enzyme conservation and the ease of phenotyping mitochondrial defects in yeast. Yet the mature sequences of all its mitochondrially-encoded tRNAs (mt-tRNAs), including the positions of chemically modified bases, have not been determined in their entirety. We used Nanopore direct RNA sequencing (DRS) of mitochondrially-enriched RNA combined with enzyme knockouts to map base modifications across 24 mt-tRNA isoacceptors in yeast. Analysis of mt-tRNAs from wild-type yeast revealed base miscalls that were coincident with known modification sites at annotated positions, as well as at previously undescribed sites. Comparison to yeast lacking the gene, which encodes a conserved pseudouridine synthase, demonstrated that 23 of 24 mt-tRNAs are pseudouridylated by Pus4 to form Ψ in the T-loop. We also mapped the uridines modified by Pus2, catalyzing Ψ and/or Ψ on several mt-tRNAs, a function matching its paralog Pus1 that modifies cytosolic tRNAs at the same positions. In yeast lacking the gene, some mt-tRNAs also showed changes at other positions inconsistent with being Pus2-pseudouridylated sites. These include U that is modified to cmnm s U in the anticodon of mt-tRNA , and A in mt-tRNA that is not previously known to be modified. Sequencing of a strain lacking the dihydrouridine synthase gene also permitted determination of the dihydrouridine sites catalyzed by Dus2. Loss of this D modification also led to pleiotropic effects on other putative modification sites, those presumed catalyzed by Dus1 and Trm1, in many mt-tRNAs. In summary these data provide a comprehensive analysis of mt-tRNA sequences, including modifications. They also suggest previously unknown RNA modification "circuits" in mt-tRNAs, in which loss of Pus2-catalyzed Ψ /Ψ and Dus2-catalyzed D causes increases or decreases in other modifications. Changes of multiple different modifications from loss of the activity of a single enzyme has implications for how mutations in these genes may cause pleiotropic effects on tRNA structure and translation.
tRNA修饰酶的突变与人类严重的线粒体疾病相关。由于酶的保守性以及在酵母中对线粒体缺陷进行表型分析的简便性，酵母已被用于模拟这些突变的影响。然而，其所有线粒体编码的tRNA（mt-tRNA）的成熟序列，包括化学修饰碱基的位置，尚未完全确定。我们使用线粒体富集RNA的纳米孔直接RNA测序（DRS）结合酶敲除技术，绘制了酵母中24种mt-tRNA同工受体上的碱基修饰图谱。对野生型酵母的mt-tRNA分析揭示了碱基错配，这些错配与注释位置以及先前未描述位置的已知修饰位点一致。与缺乏编码保守假尿苷合酶基因的酵母进行比较，结果表明24种mt-tRNA中的23种被Pus4假尿苷化，在T环中形成Ψ。我们还绘制了由Pus2催化修饰的尿苷，Pus2在几种mt-tRNA上催化Ψ和/或Ψ，其功能与其在相同位置修饰胞质tRNA的旁系同源物Pus1相匹配。在缺乏基因的酵母中，一些mt-tRNA在其他位置也出现了与Pus2假尿苷化位点不一致的变化。这些变化包括mt-tRNA反密码子中的U被修饰为cmnm5s2U，以及mt-tRNA中先前未知被修饰的A。对缺乏二氢尿苷合酶基因的菌株进行测序，也确定了由Dus2催化的二氢尿苷位点。这种D修饰的缺失也导致许多mt-tRNA中对其他假定修饰位点产生多效性影响，这些位点推测由Dus1和Trm1催化。总之，这些数据提供了对mt-tRNA序列（包括修饰）的全面分析。它们还表明mt-tRNA中存在先前未知的RNA修饰“回路”，其中Pus2催化的Ψ/Ψ和Dus2催化的D缺失会导致其他修饰的增加或减少。单一酶活性丧失导致多种不同修饰的变化，这对这些基因中的突变如何对tRNA结构和翻译产生多效性影响具有重要意义。
