Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes

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.

Kinetic mechanism of human mitochondrial RNase P

“…A control experiment demonstrates that MRPP3 does not elute in the absence of His6-MRPP1/2 when pre-tRNA is present (Lane 12). These data reveal an RNA-mediated MRPP1/2/3•pre-tRNA quaternary complex consistent with a recent report by Oerum et al (29).…”

“…If a stable MRPP1/2/3 complex forms, MRPP3 will elute from the resin along with His6-MRPP1/2. Under near-physiological salt conditions (100 mM NaCl), a MRPP1/2/3 complex was not observed, consistent with previous observations (Supplementary Figure S1) (13,29). Even at high MRPP1/2 and MRPP3 concentrations (2.1 μM), the His6-MRPP1/2/3 complex is not observed, indicating a weak affinity of MRPP3 for His6-MRPP1/2 in the absence of pre-tRNA (Lane 3).…”

“…Our data are consistent with structural studies reporting the MRPP1/2/3-tRNA complex and a distorted MRPP3 active site (25,26,29) and provide insights into the kinetics and thermodynamics of these interactions. Pre-tRNA stabilizes the MRPP1/2/3 complex as demonstrated by magnetic capture (Figure 3) and binding data (Figure 4, Table 2).…”

“…The simplest model for pre-tRNA cleavage catalyzed by human mtRNase P involves the formation of a tertiary protein complex (MRPP1/2/3), followed by the binding and cleavage of pre-tRNA (Scheme 1). However, the data presented in this work and others (13,18,25,26,29) show that MRPP3 does not strongly associate with MRPP1/2 under physiological conditions (13) and pre-tRNA binds to both MRPP1/2 and MRPP3 subunits ( Figure 4, Table 2). Furthermore, synergistic binding between MRPP1/2/3 and pre-tRNA is observed ( Table 2) and could possibly be explained by the stabilization of the MRPP1/2/3 complex by pre-tRNA ( Figure 3).…”

“…We chose this substrate so we can compare the kinetics of MRPP1/2/3 with PRORP1. STO kinetics and MTO activity with only a single time-point have been previously described for human mtRNase P (13,14,23,24,29). Here, we present the first detailed MTO kinetic analysis.…”