Abstract
Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5′-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5′-end cleavage comprises the methyltransferase domain–containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17β-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1–MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer–monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1–MRPP2–MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1–MRPP2 and MRPP1–MRPP2–MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.
Highlights
Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function
This study proposes low-resolution models of the mitochondrial RNase P protein 1 (MRPP1)– MRPP2 and MRPP1–MRPP2–MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates
MRPP1⌬mitochondrial targeting signal (MTS) was further assessed using size-exclusion chromatography– coupled multiangle light scattering (SEC-MALS) that determined an apparent mass of 56 Ϯ 1.12 kDa (Fig. 1B)
Summary
MRPP1 harbors an N-terminal dimerization domain and a C-terminal methyltransferase domain. Co-expressed MRPP1⌬MTS–MRPP2 complex, but not MRPP1⌬MTS or MRPP1MT(⌬202) alone, methylates pre-(mt)tRNAIle and pre-(mt)tRNAVal (Fig. 4D) Both m1G9 and m1A9 MTase activities of the complex are abolished with the D314N or Q226A substitution on MRPP1 (Fig. 4D), confirming the essential catalytic roles of the strictly invariant Asp-314 and Gln-226 residues. The presence of processed (mt)tRNA and 5Ј-leader by-product was observed in the reaction lane containing all three proteins together with the pre(mt)tRNA substrate (Fig. 5D, lane 3), confirming RNase P activity for the reconstituted ternary complex. We measured scattering data for MRPP3⌬206 protein, which shows a good fit (2 ϭ 1.74 for q values between 0.01 and 0.45°) to the structure (Fig. S8C) Both MRPP3⌬MTS and MRPP3⌬206 envelopes indicate a monomeric protein that is supported by analytical SEC (Fig. S4F) and in line with MRPP3 orthologues from A. thaliana (PRORP1 and PRORP2) [42]. I(0), forward scattering; Rg, radius of gyration; a.u., arbitrary units; RS-Rg, real-space radius of gyration; Porod V, Porod volume; Dmax, maximal intraparticle dimension. a.u., absorbance unit
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