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.

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.

Selective and competitive functions of the AAR and UPR pathways in stress-induced angiogenesis.

The amino acid response (AAR) and unfolded protein response (UPR) pathways converge on eIF2α phosphorylation, which is catalyzed by Gcn2 and Perk, respectively, under different stresses. This close interconnection makes it difficult to specify different functions of AAR and UPR. Here, we generated a zebrafish model in which loss of threonyl-tRNA synthetase (Tars) induces angiogenesis dependent on Tars aminoacylation activity. Comparative transcriptome analysis of the tars-mutant and wild-type embryos with/without Gcn2- or Perk-inhibition reveals that only Gcn2-mediated AAR is activated in the tars-mutants, whereas Perk functions predominantly in normal development. Mechanistic analysis shows that, while a considerable amount of eIF2α is normally phosphorylated by Perk, the loss of Tars causes an accumulation of uncharged tRNAThr, which in turn activates Gcn2, leading to phosphorylation of an extra amount of eIF2α. The partial switchover of kinases for eIF2α largely overwhelms the functions of Perk in normal development. Interestingly, although inhibition of Gcn2 and Perk in this stress condition both can reduce the eIF2α phosphorylation levels, their functional consequences in the regulation of target genes and in the rescue of the angiogenic phenotypes are dramatically different. Indeed, genetic and pharmacological manipulations of these pathways validate that the Gcn2-mediated AAR, but not the Perk-mediated UPR, is required for tars-deficiency induced angiogenesis. Thus, the interconnected AAR and UPR pathways differentially regulate angiogenesis through selective functions and mutual competitions, reflecting the specificity and efficiency of multiple stress response pathways that evolve integrally to enable an organism to sense/respond precisely to various types of stresses.

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys.

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

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.

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.

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.