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.
Highlights
Transfer RNAs are the essential adaptors in the decoding of messenger RNAs by the ribosome; their faithful biogenesis is crucial for cell survival. tRNAs are transcribed as precursors and undergo several steps of nucleolytic processing, nucleotide addition, editing and nucleotide modification [1]. tRNAs are the most extensively modified type of cellular RNA, and to date, $100 different modifications have been identified [2,3]
A corresponding enzymatic activity was previously found in human mitochondrial extracts [26]. We investigated whether this activity is owned by tRNA methyltransferase 10 C (TRMT10C) and, is associated with mtRNase P
We used atRNAIle (Figure 1D) with all guanosines labelled as a substrate, and crude HeLa cell mitochondrial extracts or tagged recombinant proteins purified by affinity chromatography (Supplementary Figure S1) for in vitro methylation assays; the RNA was subsequently isolated, hydrolysed and 50 nucleoside monophosphates resolved by thin-layer chromatography (TLC) (Figure 2A)
Summary
Transfer RNAs (tRNAs) are the essential adaptors in the decoding of messenger RNAs (mRNAs) by the ribosome; their faithful biogenesis is crucial for cell survival. tRNAs are transcribed as precursors and undergo several steps of nucleolytic processing, nucleotide addition, editing and nucleotide modification [1]. tRNAs are the most extensively modified type of cellular RNA, and to date, $100 different modifications have been identified [2,3]. Transfer RNAs (tRNAs) are the essential adaptors in the decoding of messenger RNAs (mRNAs) by the ribosome; their faithful biogenesis is crucial for cell survival. TRNAs are the most extensively modified type of cellular RNA, and to date, $100 different modifications have been identified [2,3]. The effects of modifications in the tRNA core are less obvious and still poorly understood. Most core-modifications are pseudouridinylations and different kinds of methylations at several nucleotide positions, and they are thought to stabilize the 3D structure of tRNAs by favouring or impeding specific base interactions [4,5]. The lack of a single of these modifications often has unremarkable effects, but the removal of more than one can structurally and metabolically destabilize tRNAs, and thereby preclude cell survival [6]
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