Complete chemical structures of human mitochondrial tRNAs

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications

S-Adenosylmethionine Synthesis Is Regulated by Selective N6-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1

Summary: S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N6-methyladenosine (m6A) in the 3′ UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3′ UTR. Knockdown of METTL16 and the m6A reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3′ UTR revealed that these m6As were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels. : Shima et al. find that MAT2A mRNA is stabilized upon depletion of intracellular S-adenosylmethionine (SAM). This regulation involves m6A modification in the 3′ UTR, the m6A writer METTL16, and the reader YTHDC1. Additionally, the authors show that multiple specific sites in hairpin regions of the 3′ UTR are targeted by METTL16. Keywords: cycloleucine, MAT2A, methionine adenosyltransferase, METTL16, methyladenosine, RNA, RNA degradation, S-adenosylmethionine, untranslated region, YTHDC

Specificity, synergy, and mechanisms of splice-modifying drugs

Abstract Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce mechanistically interpretable quantitative models for the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we obtain quantitative models for two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively characterize the specificities of risdiplam and branaplam for 5’ splice site sequences, suggest that branaplam recognizes 5’ splice sites via two distinct interaction modes, and contradict the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. The results also show that anomalous single-drug cooperativity, as well as multi-drug synergy, are widespread among small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our quantitative models thus clarify the mechanisms of existing treatments and provide a basis for the rational development of new therapies

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

<p>Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates</p

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

<div><p>The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m¹A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m¹A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m¹A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited <i>Escherichia coli</i> showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.</p></div

TRMT61B is responsible for m¹A947 in mitochondrial 16S rRNA.

<p>(A) Detection of hypomodified m¹A947 in mitochondrial 16S rRNA from siRNA-treated HeLa cells. After knockdown of luciferase (si-Luc, control), TRMT61B, or TRMT10C mRNAs, primer extension was used to detect methylated or non-methylated A947. The cells were transfected twice with siRNA and harvested 4 days after the first transfection. The knockdown efficiencies of TRMT61B and TRMT10C mRNAs were quantified by qRT-PCR and normalized to ACTB mRNA. The steady-state levels of both mRNAs were decreased to 5.7% compared to the mock cells. The primers are shown as solid lines next to the rRNA or tRNA, and nascent cDNAs synthesized from the primers are depicted as gray lines. (B) Detection of hypomodified m¹A58 in mitochondrial tRNALeu^(UUR) to confirm TRMT61B knockdown. (C) Nucleotide frequencies in cDNA reads corresponding to position 947 in human mitochondrial 16S rRNA. RNA-seq reads of total RNAs from HeLa cells treated with siRNAs targeting luciferase (control) or TRMT61B were mapped against the human mtDNA sequence. Nucleotides frequencies (%) were calculated from the total read coverage. Exact values are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002557#pbio.1002557.s001" target="_blank">S1 Data</a>. (D) In vitro reconstitution of m¹A947 with recombinant TRMT61B in the presence of AdoMet. Total RNAs from HeLa cells treated with siRNAs for TRMT61B (si-TRMT61B) were incubated with recombinant TRMT61B in the presence or absence of Ado-Met. m¹A947 formation in mitochondrial 16S rRNA was detected by primer extension. Total RNAs of si-Luc and si-TRMT61B were used as controls for primer extension. (E) In vitro reconstitution of m¹A947 with wild-type TRMT61B and its active-site D335A mutant. The 114-mer RNA segment including Helix 71 (G866-U979) of human mitochondrial 16S rRNA was incubated with wild-type TRMT61B or its D335A mutant in the presence or absence of Ado-Met, followed by RNase A digestion, and subjected to LC/MS analysis. Mass-spec chromatograms detect doubly-charged negative ions of the tetramer fragment carrying m¹A947 (upper panels, positions 945–948, m/z 661.60, and MW 1325.21) and the corresponding unmodified fragment (lower panels, m/z 654.59, MW 1311.19).</p

High structural conservation between <i>S</i>. <i>scrofa</i> mitoribosome and <i>E</i>. <i>coli</i> ribosome at position 947.

<p>(<b>A</b>) The structure of the porcine mitoribosomal large subunit (PDB accession code 4v1a and 4v19) shown from the subunit interface side. The ribosomal RNA is shown in brown and the ribosomal proteins in green. The ribosomal tRNA A-, P-, and E-binding sites are indicated. (<b>B</b>) Sticks-and-ribbon representation of interaction between helices H71 and H64 in <i>S</i>. <i>scrofa</i> (brown) mitoribosome or <i>E</i>. <i>coli</i> (turquoise) ribosome (PDB accession code 4ybb). The hydrogen bond that is likely disrupted by an adenine in position 947 is represented as a dashed line. (<b>C</b>) The positively charged m¹A947 stabilizes the structure by interacting with the negatively charged H64 backbone. Numbers refer to the positions of <i>E</i>. <i>coli</i> ribosomal RNA.</p

Nucleotide distribution in reads corresponding to 16S rRNA position 947 in humans.

<p>(<b>A</b>) RNA-Seq reads from nine species were mapped to their corresponding mtDNA sequence. Notice that species with an adenine in their mtDNA exhibit the RDDs, while species with a thymine do not (species 8–9). Total read coverage of all samples is shown above each species. (<b>B</b>) Coverage and nucleotide distribution of deep sequencing of <i>Sus scrofa</i> samples at the orthologue of human 16S rRNA position 947. The right histogram trio stem from an isolated ribosome (enriched for mature rRNA), and the middle trio stem from total mitochondrial RNA (which contains a mixture of mature and premature rRNAs). The left-most trio of histograms stem from mitochondrial DNA. Notice that the level of m¹A modification increased in the right trio as compared to the middle one. Exact values are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002557#pbio.1002557.s001" target="_blank">S1 Data</a>.</p

Human mitochondrial 16S rRNA is methylated at position 947.

<p>(<b>A</b>) Capillary LC/ESI-MS analysis of RNA fragments of human mitochondrial 16S rRNA digested with RNase T₁. The upper panel shows a base-peak chromatogram (BPC), and the lower panel represents mass chromatogram for detecting quintuple (-5)-charged ion of the methylated 12-mer fragment (UUCCUUAAm¹AUAGp, m/z 765.7). (<b>B</b>) Collison-induced dissociation spectrum of the methylated 12-mer fragment. The sequence was confirmed by assignment of the product ions. Nomenclature for the product ions is in accordance with a previous report [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002557#pbio.1002557.ref022" target="_blank">22</a>].</p