Structural and functional basis of mitochondrial tRNA processing

The mammalian mitochondria are subcellular organelles, generating energy in the cell by the synthesis of adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS). The maintenance of proper mitochondrial (mt) structure and function requires more than 1000 proteins generated from the nuclear DNA (nDNA) expression and 13 protein subunits generated from the mitochondrial DNA (mtDNA) expression. The transcription of the two strands of mtDNA generates two long polycistronic precursor ribonucleic acid (pre-RNA) molecules harboring the precursor messenger RNAs (pre-mRNAs), precursor ribosomal RNAs (pre-rRNAs) and precursor transfer RNAs (pre-tRNAs) at a stretch. The processing of pre-tRNAs punctuating the pre-rRNAs and most of the pre-mRNAs causes the release of each of the RNA species that undergo their own respective maturation pathways to attain the functional state. This thesis explores the molecular mechanism of pre-tRNA processing in human mitochondria, critical for several downstream processes such as mitoribosome biogenesis and the maturation of mt-mRNAs and mt-tRNAs. The first step of pre-tRNA processing is performed by the human mitochondrial ribonuclease P (mt-RNase P) composed of three protein subunits: MRPP1, MRPP2 and MRPP3 that excises the tRNA at the 5 ́-end. In Paper I, the high-resolution crystal structure o the MRPP3 protein is described. We observed that the MRPP3 protein is unable to perform 5 ́- tRNA cleavage on its own because of its distorted active site. Unlike its structural homologue: protein-only RNase P 1 (PRORP1) found in the plant mitochondria and chloroplasts, MRPP3 requires a subcomplex of MRPP1 and MRPP2 (MRPP1/2) and pre- tRNA substrate to undergo conformational changes in the presence of metal ions to achieve an active state. In Paper II, previously unknown central role of MRPP1/2 complex as a maturation platform for mt-tRNAs is reported. We show that the MRPP1/2 complex is not just an essential component of human mt-RNase P, but also significantly enhances the 3 ́- processing by the ELAC2 protein in 17 out of 22 5 ́-processed mt-tRNAs. Moreover, the CCA addition to the 3 ́-processed tRNAs was shown to be possible while the tRNA remained bound to the MRPP1/2 complex. Thus, the MRPP1/2 sub-complex hosts the pre-tRNA substrate through three major steps of the processing activities instead of just one as thought previously. The human mt-tRNA genes in the mtDNA are locations for several disease-causing mutations causing mt encephalomyopathies. In Paper III, the role of disease-causing tRNA(Lys) acceptor stem mutations is evaluated in terms of its effects on mt-RNase P activity. An overall impairment of RNase P processing was observed in tRNA acceptor stem mutants in relation to the wild type tRNA. The MRPP1/2 complex can bypass a single point mutation in the acceptor stem of substrate pre-tRNA and form a stable complex in vitro. However, the nuclease activity by MRPP3 is strongly affected. We speculate that either the MRPP3 protein is unable to form a complex with MRPP1/2-pre-tRNA or the re-organization of its active site upon binding to MRPP1/2-pre-tRNA is affected as a consequence of the acceptor stem mutation