Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria.
Institute of Pharmaceutical Chemistry, Philipps-University Marburg, 35037 Marburg, Germany.
Nucleic Acids Res. 2023 Oct 27;51(19):10536-10550. doi: 10.1093/nar/gkad713.
RNase P is the endonuclease responsible for the 5' processing of precursor tRNAs (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 including 12 different mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORP's PPR domain, appears basically retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORP's nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. This functional dependence of human PRORP on an extra tRNA-binding protein complex likely reflects an evolutionary adaptation to the erosion of canonical structural features in mitochondrial tRNAs.
RNase P 是负责前体 tRNA（pre-tRNA）5'加工的内切核酸酶。与植物或原生生物中发现的单一亚基蛋白-only RNase P（PRORP）不同，人线粒体 RNase P 是一种多酶组装体，除了同源的 PRORP 亚基外，还包含一个甲基转移酶（TRMT10C）和一个脱氢酶（SDR5C1）亚基；这些蛋白质（但不是它们的酶活性）是有效切割 pre-tRNA 所必需的。在这里，我们报告了人 PRORP 的切割反应的动力学分析及其与 TRMT10C-SDR5C1 的相互作用，包括 12 种不同的线粒体 pre-tRNA。令人惊讶的是，我们发现 PRORP 本身以纳摩尔亲和力结合 pre-tRNA，甚至在没有其他亚基的情况下以降低的效率也能切割其中一些。因此，涉及 tRNA 弯曲和 PRORP 的 PPR 结构域的古老结合模式似乎基本上被人 PRORP 保留，其金属核酸酶结构域在原则上是正确折叠和有功能的。我们的发现支持了这样一种模型，即 TRMT10C-SDR5C1 的主要功能是将 PRORP 的核酸酶结构域引导到切割位点，从而提高切割的速度和准确性。人 PRORP 对额外的 tRNA 结合蛋白复合物的功能依赖性可能反映了对线粒体 tRNA 中典型结构特征侵蚀的进化适应。
