Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA.
Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada.
Nucleic Acids Res. 2020 Aug 20;48(14):7924-7943. doi: 10.1093/nar/gkaa592.
Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.
哺乳动物线粒体核糖体（mitoribosomes）的生物发生涉及几种保守的小 GTP 酶。在这里，我们报告说 Obg 家族蛋白 GTPBP5 或 MTG2 是一种线粒体蛋白，其在 TALEN 诱导的 HEK293T 敲除（KO）细胞系中的缺失导致 55S 单体的水平严重降低，并减弱了线粒体蛋白的合成。我们表明，GTPBP5 的一部分与大亚基（mtLSU）共沉降，并与 16S rRNA 以及几种 mtLSU 蛋白和组装因子特异性交联。值得注意的是，后者包括参与单体组装的 MTERF4 和催化 16S mt-rRNA A 环 U1369 残基甲基化修饰的 MRM2 甲基转移酶。使用邻近依赖性生物素化（BioID）测定还检测到 GTPBP5 与 MRM2 的相互作用。在 GTPBP5-KO 线粒体中，mtLSU 缺乏 bL36m，积累了过量的组装因子 MTG1、GTPBP10、MALSU1 和 MTERF4，并含有低甲基化的 16S rRNA。我们提出，GTPBP5 主要通过确保两个 16S rRNA 残基的有效甲基化来为 mtLSU 的成熟提供动力，并最终通过释放晚期 mtLSU 组装因子来协调亚基连接。通过这种方式，GTPBP5 在 mtLSU 组装过程中提供了最终的质量控制检查点功能，最大限度地减少了过早的亚基连接，以确保成熟的 55S 单体的组装。
