A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Abstract

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m1G9 and m1A9 formation. The ability of the mitochondrial tRNA:m1R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.

A novelHSD17B10mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

“…1C), which form 2 helices (residues 204-208 and residues 211-217) separated by a sharp turn. 32 K212 is in the second helix acting as the "lid" to close the active-site [17][18][19][20][21][22][23], the insertion segments for subunit-subunit interaction (residues 100-110 and residues 141-146), the catalytic triad for the dehydrogenase (S155, Y168, and K172), and the substrate-specificity loop (residues 203-220) 32 . (B) The position of p.K212E in the tetramer structure of human SDR5C1.…”

“…We chose to determine the mutational effect by comparing known quantities of purified wildtype and mutant enzyme, each expressed and purified from an E. coli over-expression strain. 19,34 The assay used acetoacetylCoA (3-ketoacyl-CoA) as the substrate and monitored its reduction to 3-hydroxyacetyl-CoA accompanied by oxidation of NADH to NAD C . While the wild-type enzyme had an activity of 3.18 § 0.18 units/mg, similar to other reported values, 34 the mutant showed an almost 4-fold decrease of the activity to 0.84 § 0.12 units/mg (Fig.…”

“…7 We next determined whether the p.K212E mutation decreases the methyl transferase activity of TRMT10C, which requires the presence of SDR5C1 to be active. 19,34 We assayed a reconstituted and recombinant form of the TRMT10C-SDR5C sub-complex with AdoMet as the methyl donor and the N 1 of G9 in the unmodified transcript of mt-tRNA Leu (UUR) as the acceptor (Fig. 2B).…”

“…19,34 Protein concentration was determined by Bradford assay relative to bovine serum albumin standards and adjusted by image analysis of purity on SDS-PAGE. The mutation p.K212E was introduced into the plasmid-encoded recombinant HSD17B10 gene by site-directed mutagenesis and verified by sequence analysis.…”

“…18 SDR5C1 (MRPP2) forms a sub-complex with TRMT10C (MRPP1), a mitochondrial tRNA methyl transferase that synthesizes the methylated m 1 G9 or m 1 A9 on mt-tRNAs. 19 This methylation is crucial for proper folding of mt-tRNA Lys 20 and may be important for folding of 19 of the 22 mt-tRNAs that contain purines at position 9. The sub-complex forms a ternary complex with PRORP (MRPP3), which cleaves polycistronic mitochondrial transcripts to generate 5 0 -processed mt-tRNAs.…”