Structural basis of human mitochondrial RNA processing
by Arjun Bhatta
Date of Examination:2024-08-08
Date of issue:2024-12-16
Advisor:Prof. Dr. Hauke Hillen
Referee:Prof. Dr. Kai Heimel
Referee:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Kai Tittmann
Persistent Address:
http://dx.doi.org/10.53846/goediss-10923
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Abstract
English
Mitochondrial transcription produces polycistronic transcripts, in which ribosomal RNA (rRNA) and messenger RNA (mRNA) regions are interspersed with transfer RNA (tRNA) sequences. Endonucleolytic cleavages at the 5' and 3' ends of tRNA sequences in polycistronic transcripts are the first critical post-transcriptional steps in mitochondrial gene expression, essential for the release of all functional rRNA, mRNA, and tRNA species. Mitochondrial tRNA 5’ processing is catalyzed by the mitochondrial RNase P (mt-RNase P) complex composed of an endonuclease subunit, PRORP, and two auxiliary subunits forming a tRNA methyltransferase subcomplex, TRMT10C and SDR5C1. As a multi-subunit protein-only RNase P, the human mt-RNase P complex is distinct from the ubiquitous ribonucleoprotein RNase Ps or single-subunit protein-only RNase Ps. The 3’ processing of both mitochondrial and nuclear tRNAs is catalyzed by a common RNase Z enzyme, ELAC2. Recent evidence indicates that ELAC2 may also depend on TRMT10C and SDR5C1 specifically for mitochondrial tRNA processing. Thus, mitochondrial RNases P and Z appear to be compositionally atypical multimeric enzymes, and the molecular underpinnings of their function are not well understood. This dissertation focuses on structural and mechanistic characterization of human mt-RNase P and Z complexes. First, to understand how mt-RNase P complex carries out tRNA 5’ processing and what role TRMT10C and SDR5C1 play therein, we determined a cryo-electron microscopy structure of human mt-RNase P bound to precursor tRNA. The structure reveals that the subunits TRMT10C and SDR5C1 form a subcomplex that recognizes tRNAs via a sequence-independent mechanism, enabling the recognition of structurally diverse mt-tRNAs. Binding to TRMT10C–SDR5C1 subcomplex distorts the tRNA structure and positions the 9th nucleobase in the methyltransferase active site. The endonuclease PRORP is recruited via interactions with TRMT10C, which stabilize PRORP in an active conformation and ensure precise cleavage at the 5' end of the pre-tRNA. Subsequently, we investigated the mechanism of tRNA 3’ processing and the role of TRMT10C and SDR5C1 in this process through a combination of structural and biochemical studies. We found that TRMT10C and SDR5C1 are specifically required for processing of mt-tRNAs lacking the canonical elbow regions. ELAC2 recognizes nuclear and mitochondrial tRNAs with canonical elbows via direct interactions with the conserved elbow elements. By contrast, most mt-tRNAs, which lack a canonical elbow, depend on compensatory protein-protein interactions between ELAC2 and TRMT10C for processing. Together, these results elucidate the structural and mechanistic basis of the mitochondrial tRNA 5’ and 3’ processing, provide catalytic insights into tRNA-R9 methylation and explain the role of TRMT10C and SDR5C1 in mt-tRNA processing. Furthermore, they highlight the role of TRMT10C–SDR5C1 complex as a general mt-tRNA maturation platform that functions as a mitochondrial tRNA ‘prosthesis’, stabilizing the tertiary fold of mt-tRNAs and compensating for the absence of conserved structural features otherwise necessary for their recognition. This work represents an important milestone towards a complete structural understanding of mitochondrial gene expression and provides a framework for future studies aimed at achieving this goal.
Keywords: Mitochondrial gene expression; Ribonuclease P; Ribonuclease Z; PRORP; ELAC2; Cryo electron microscopy; tRNA processing; tRNA maturation; TRMT10C; tRNA methylation
