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.

Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein, RBP, which facilitates host-cell attachment and plays a fundamental role in determining host-range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently definedNarmovirusgenus within theParamyxoviridaefamily. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed β-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed β-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs.