A dual role of human tRNA methyltransferase hTrmt13 in regulating translation and transcription.

Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA-binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2'-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial-mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA-modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.

Multifaceted roles of t6A biogenesis in efficiency and fidelity of mitochondrial gene expression.

N 6-Threonylcarbamoyladenosine at A37 (t6A37) of ANN-decoding transfer RNAs (tRNAs) is a universal modification whose functions have been well documented in bacteria and lower eukaryotes; however, its role in organellar translation is not completely understood. In this study, we deleted the mitochondrial t6A37-modifying enzyme OSGEPL1 in HEK293T cells. OSGEPL1 is dispensable for cell viability. t6A37 hypomodification selectively stimulated N1-methyladenosine at A9 (m1A9) and N2-methylguanosine at G10 (m2G10) modifications and caused a substantial reduction in the aminoacylation of mitochondrial tRNAThr and tRNALys, resulting in impaired translation efficiency. Multiple types of amino acid misincorporation due to the misreading of near-cognate codons by t6A37-unmodified tRNAs were detected, indicating a triggered translational infidelity. Accordingly, the alterations in mitochondrial structure, function, and the activated mitochondrial unfolded protein response were observed. Mitochondrial function was efficiently restored by wild-type, but not by tRNA-binding-defective OSGEPL1. Lastly, in Osgepl1 deletion mice, disruption to mitochondrial translation was evident but resulted in no observable deficiency under physiological conditions in heart, which displays the highest Osgepl1 expression. Taken together, our data delineate the multifaceted roles of mitochondrial t6A37 modification in translation efficiency and quality control in mitochondria.

Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Loss of threonyl-tRNA synthetase-like protein Tarsl2 has little impact on protein synthesis but affects mouse development.

Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly, TARSL2 is a recently evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activities in vitro, whether it is a true tRNA synthetase for mRNA translation in vivo is unclear. In this study, we showed that Tars1 is an essential gene since homozygous Tars1 KO mice were lethal. In contrast, when Tarsl2 was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied on Tars1 but not on Tarsl2 for mRNA translation. Furthermore, Tarsl2 deletion did not influence the integrity of the multiple tRNA synthetase complex, suggesting that Tarsl2 is a peripheral member of the multiple tRNA synthetase complex. Finally, we observed that Tarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss of Tarsl2 has little influence on protein synthesis but does affect mouse development.

Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs.

N 6-Threonylcarbamoyladenosine (t6A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t6A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t6A moiety. Using t6A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNAMet and modification enzymes. The role of the universal CCA end in t6A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNAIle aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t6A functions as a tRNAIle isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t6A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t6A in tRNAIle aminoacylation and codon decoding in human cells.

RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.

Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.

Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8.

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m3C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m3C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m3C32 modification of human mitochondrial tRNAThr (hmtRNAThr) was not reliant on t6A modification at A37 (t6A37), while that of hmtRNASer(UCN) critically depended on i6A modification at A37 (i6A37). We clarified the hmtRNAThr substrate recognition mechanism, which was obviously different from that of hmtRNASer(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m3C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m3C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.

Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2.

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.

The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases.

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.

Intellectual disability-associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs.

tRNA modifications at the anti-codon loop are critical for accurate decoding. FTSJ1 was hypothesized to be a human tRNA 2'-O-methyltransferase. tRNAPhe (GAA) from intellectual disability patients with mutations in ftsj1 lacks 2'-O-methylation at C32 and G34 (Cm32 and Gm34). However, the catalytic activity, RNA substrates, and pathogenic mechanism of FTSJ1 remain unknown, owing, in part, to the difficulty in reconstituting enzymatic activity in vitro. Here, we identify an interacting protein of FTSJ1, WDR6. For the first time, we reconstitute the 2'-O-methylation activity of the FTSJ1-WDR6 complex in vitro, which occurs at position 34 of specific tRNAs with m1 G37 as a prerequisite. We find that modifications at positions 32, 34, and 37 are interdependent and occur in a hierarchical order in vivo. We also show that the translation efficiency of the UUU codon, but not the UUC codon decoded by tRNAPhe (GAA), is reduced in ftsj1 knockout cells. Bioinformatics analysis reveals that almost 40% of the high TTT-biased genes are related to brain/nervous functions. Our data potentially enhance our understanding of the relationship between FTSJ1 and nervous system development.

Modifications of the human tRNA anticodon loop and their associations with genetic diseases.

Transfer RNAs (tRNAs) harbor the most diverse posttranscriptional modifications. Among such modifications, those in the anticodon loop, either on nucleosides or base groups, compose over half of the identified posttranscriptional modifications. The derivatives of modified nucleotides and the crosstalk of different chemical modifications further add to the structural and functional complexity of tRNAs. These modifications play critical roles in maintaining anticodon loop conformation, wobble base pairing, efficient aminoacylation, and translation speed and fidelity as well as mediating various responses to different stress conditions. Posttranscriptional modifications of tRNA are catalyzed mainly by enzymes and/or cofactors encoded by nuclear genes, whose mutations are firmly connected with diverse human diseases involving genetic nervous system disorders and/or the onset of multisystem failure. In this review, we summarize recent studies about the mechanisms of tRNA modifications occurring at tRNA anticodon loops. In addition, the pathogenesis of related disease-causing mutations at these genes is briefly described.

Molecular basis for t6A modification in human mitochondria.

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

Nitrosative stress inhibits aminoacylation and editing activities of mitochondrial threonyl-tRNA synthetase by S-nitrosation.

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.

Molecular basis of the multifaceted functions of human leucyl-tRNA synthetase in protein synthesis and beyond.

Human cytosolic leucyl-tRNA synthetase (hcLRS) is an essential and multifunctional enzyme. Its canonical function is to catalyze the covalent ligation of leucine to tRNALeu, and it may also hydrolyze mischarged tRNAs through an editing mechanism. Together with eight other aminoacyl-tRNA synthetases (AaRSs) and three auxiliary proteins, it forms a large multi-synthetase complex (MSC). Beyond its role in translation, hcLRS has an important moonlight function as a leucine sensor in the rapamycin complex 1 (mTORC1) pathway. Since this pathway is active in cancer development, hcLRS is a potential target for anti-tumor drug development. Moreover, LRS from pathogenic microbes are proven drug targets for developing antibiotics, which however should not inhibit hcLRS. Here we present the crystal structure of hcLRS at a 2.5 Å resolution, the first complete structure of a eukaryotic LRS, and analyze the binding of various compounds that target different sites of hcLRS. We also deduce the assembly mechanism of hcLRS into the MSC through reconstitution of the entire mega complex in vitro. Overall, our study provides the molecular basis for understanding both the multifaceted functions of hcLRS and for drug development targeting these functions.

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor.

Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

Newly acquired N-terminal extension targets threonyl-tRNA synthetase-like protein into the multiple tRNA synthetase complex.

A typical feature of eukaryotic aminoacyl-tRNA synthetases (aaRSs) is the evolutionary gain of domains at either the N- or C-terminus, which frequently mediating protein-protein interaction. TARSL2 (mouse Tarsl2), encoding a threonyl-tRNA synthetase-like protein (ThrRS-L), is a recently identified aaRS-duplicated gene in higher eukaryotes, with canonical functions in vitro, which exhibits a different N-terminal extension (N-extension) from TARS (encoding ThrRS). We found the first half of the N-extension of human ThrRS-L (hThrRS-L) is homologous to that of human arginyl-tRNA synthetase. Using the N-extension as a probe in a yeast two-hybrid screening, AIMP1/p43 was identified as an interactor with hThrRS-L. We showed that ThrRS-L is a novel component of the mammalian multiple tRNA synthetase complex (MSC), and is reliant on two leucine zippers in the N-extension for MSC-incorporation in humans, and mouse cell lines and muscle tissue. The N-extension was sufficient to target a foreign protein into the MSC. The results from a Tarsl2-deleted cell line showed that it does not mediate MSC integrity. The effect of phosphorylation at various sites of hThrRS-L on its MSC-targeting is also explored. In summary, we revealed that ThrRS-L is a bona fide component of the MSC, which is mediated by a newly evolved N-extension domain.

Archaeal NSUN6 catalyzes m5C72 modification on a wide-range of specific tRNAs.

Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m5C) at C72 of four specific tRNAs, and its homologs are present only in higher eukaryotes and hyperthermophilic archaea. Archaeal NSun6 homologs possess conserved catalytic residues, but have distinct differences in their RNA recognition motifs from eukaryotic NSun6s. Until now, the biochemical properties and functions of archaeal NSun6 homologs were unknown. In archaeon Pyrococcus horikoshii OT3, the gene encoding the NSun6 homolog is PH1991. We demonstrated that the PH1991 protein could catalyze m5C72 formation on some specific PhtRNAs in vitro and was thus named as PhNSun6. Remarkably, PhNSun6 has a much wider range of tRNA substrates than hNSun6, which was attributed to its tRNA substrate specificity. The mechanism was further elucidated using biochemical and crystallographic experiments. Structurally, the binding pocket for nucleotide 73 in PhNSun6 is specific to accommodate U73 or G73-containing PhtRNAs. Furthermore, PhNSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by PhNSun6. Functionally, the m5C72 modification could slightly promote the thermal stability of PhtRNAs, but did not affect the amino acid accepting activity of PhtRNAs.

The G3-U70-independent tRNA recognition by human mitochondrial alanyl-tRNA synthetase.

Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg663, likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.

A natural non-Watson-Crick base pair in human mitochondrial tRNAThr causes structural and functional susceptibility to local mutations.

Six pathogenic mutations have been reported in human mitochondrial tRNAThr (hmtRNAThr); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNAThr and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson-Crick base pair A29/C41 caused hmtRNAThr to be highly susceptible to mutations disrupting the G30-C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNAThr, and editing mischarged tRNAThr. A similar phenomenon was observed for hmtRNATrp with an A29/C41 non-Watson-Crick base pair, but not in bovine mtRNAThr with a natural G29-C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t6A modification at A37, which is essential for the coding properties of hmtRNAThr. In summary, our results revealed the crucial role of the G30-C40 base pair in maintaining the proper structure and function of hmtRNAThr because of A29/C41 non-Watson-Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.