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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 KARSencodes 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 vitroand in vivo.We constructed two yeast genetic models, which are powerful tools to test the in vivoaminoacylation activity of KARSmutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARSc.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARSmutations in the future.

TRMT1 is an N2-methylguanosine (m2G) and N2,N2-methylguanosine (m22G) methyltransferase that targets G26 of both cytoplasmic and mitochondrial tRNAs. In higher eukaryotes, most cytoplasmic tRNAs with G26 carry m22G26, although the majority of mitochondrial G26-containing tRNAs carry m2G26 or G26, suggesting differences in the mechanisms by which TRMT1 catalyzes modification of these tRNAs. Loss-of-function mutations of human TRMT1result in neurological disorders and completely abrogate tRNA:m22G26 formation. However, the mechanism underlying the independent catalytic activity of human TRMT1 and identity of its specific substrate remain elusive, hindering a comprehensive understanding of the pathogenesis of neurological disorders caused by TRMT1mutations. Here, we showed that human TRMT1 independently catalyzes formation of the tRNA:m2G26 or m22G26 modification in a substrate-dependent manner, which explains the distinct distribution of m2G26 and m22G26 on cytoplasmic and mitochondrial tRNAs. For human TRMT1-mediated tRNA:m22G26 formation, the semi-conserved C11:G24 serves as the determinant, and the U10:A25 or G10:C25 base pair is also required, while the size of the variable loop has no effect. We defined the requirements of this recognition mechanism as the “m22G26 criteria”. We found that the m22G26 modification occurred in almost all the higher eukaryotic tRNAs conforming to these criteria, suggesting the “m22G26 criteria” are applicable to other higher eukaryotic tRNAs.

Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5A>G, p.D2G; c.1367C>T, p.S456L; c.1535G>A, p.R512Q and c.1846_1847del, p. Y616Lfs*6 of RARS1gene, which encodes two forms of human cytoplasmic arginyl-tRNA synthetase (hArgRS), are linked to Pelizaeus-Merzbacher-like disease (PMLD) with unclear pathogenesis. Among these mutations, c.5A>G is the most extensively reported mutation, leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS. Here, we showed the detrimental effects of R512Q substitution and ΔC mutations on the structure and function of hArgRS, while the most frequent mutation c.5A>G, p.D2G acted in a different manner without impairing hArgRS activity. The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1mutations in PMLD.

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4–6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.

ABSTRACTThe apicomplexan parasites Cryptosporidiumand Toxoplasmaare serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidiumparasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidiumleucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasmaparasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidiumand Toxoplasmaby forming a covalent adduct with tRNALeuin the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

Lysosomal amino acid accumulation is implicated in several diseases, but its role in insulin resistance, the central mechanism to type 2 diabetes and many metabolic diseases, is unclear. In this study, we show the hepatic expression of lysosomal membrane protein solute carrier family 7 member 14 (SLC7A14) is increased in insulin-resistant mice. The promoting effect of SLC7A14 on insulin resistance is demonstrated by loss- and gain-of-function experiments. SLC7A14 is further demonstrated as a transporter resulting in the accumulation of lysosomal γ-aminobutyric acid (GABA), which induces insulin resistance via inhibiting mTOR complex 2 (mTORC2)’s activity. These results establish a causal link between lysosomal amino acids and insulin resistance and suggest that SLC7A14 inhibition may provide a therapeutic strategy in treating insulin resistance-related and GABA-related diseases and may provide insights into the upstream mechanisms for mTORC2, the master regulator in many important processes.

Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNALeuCAAand tRNALeuUAAisoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2′-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i6) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2′-O-methylation relies on prior N6-isopentenyladenosine modification at position 37.

This paper examines the role of amino acids connecting the aminoacylation and editing domains of leucyl-tRNA synthetase. Deletion of these amino acids resulted in complete loss of aminoacylation retention of editing activity. These results indicate that the bridge between the aminoacylation domain and editing domain is crucial for the function of the synthetase.

Abstract: Heme, as a prosthetic group of proteins, is an iron-protoporphyrin involved in a wide range of cellular functions. Cellular heme levels vary due to the accurate balance of its synthesis and degradation. The “heme sensor protein” is currently a focus of investigation because heme has been found as a cellular signaling messenger involved in various biologic processes, including gene expression, protein localization, protein stability and microRNA processing. Several eukaryotic transcriptional factors can be regulated by heme, including heme activator protein (Hap1), Bach1, REV-erbα, and neuronal PAS domain protein 2 (NPAS2). Especially, the two circadian transcriptional factors serving as the heme sensor, REV-erbα and NPAS2, coordinate the circadian clock with metabolic pathways. It is well established that heme regulates the activity of heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), which serves as a feedback inhibitor of protein translation in both erythroid and non-erythroid cells. Additionally, heme is involved in protein degradation by inducing the degradation of several proteins such as the iron response regulator (Irr), iron regulatory protein 2 (IRP2), Bach1, and circadian factor period 2 (Per2). The N-end rule ubiquitin-dependent protein degradation pathway has also been identified as a sensor of heme, which blocks the function of arginyl-tRNA protein transferase (ATE1) and E3 ubiquitin ligase. In this review, we summarize the regulatory roles of heme at the levels of transcription, protein translation, and protein degradation, highlighting the role of heme in maintaining cellular homeostasis.

Leucyl-, isoleucyl-, and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that attach similar amino acids to their cognate tRNAs. To prevent amino acid misincorporation during translation, these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that LeuRS from the deep-branching bacterium Aquifex aeolicus edits the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNA(Ile), Val-tRNA(Ile), Val-tRNA(Val), Thr-tRNA(Val), and Ile-tRNA(Leu). This unusual enlarged editing property was studied in a model of a primitive editing system containing a composite minihelix carrying the triple leucine, isoleucine, and valine identity mimicking the primitive tRNA precursor. We found that the freestanding LeuRS editing domain can edit this precursor in contrast to IleRS and ValRS editing domains. These results suggest that A. aeolicus LeuRS carries editing properties that seem more primitive than those of IleRS and ValRS. They suggest that the A. aeolicus editing domain has preserved the ambiguous editing property from the ancestral common editing domain or, alternatively, that this plasticity results from a specific metabolic adaptation.

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNAArgs with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS (argS´) was amplified by PCR and the gene encoding tRNAArg (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNAArg (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNAArgs from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys382 and Lys385 of the KMSK sequence and at Gly136 upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys385 and Gly136 for the amino acid-activation activity of B. stearothermophilus ArgRS.

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.

Escherichia coli4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher Kmvalues. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.

A simple method for preparation of α-sarcin and an antifungal protein (AFP) from the moldAspergillus giganteusMDH 18894 has been developed. α-Sarcin and AFP were purified simultaneously by carboxymethylcellulose 52 cation-exchange chromatography and Blue Sepharose CL-6B affinity chromatography. By this method, 4.2 mg of α-sarcin and 6.8 mg of AFP were obtained from 2 liters of medium. Compared with other purification methods such as gel-filtration chromatography, this procedure was simple and specific. The purified α-sarcin and AFP were homogeneous characterized on SDS–polyacrylamide gel. The enzymatic activity of several ribosome-inactivating proteins such as α-sarcin, trichosanthin, and cinnamomin was significantly inhibited by the dye Cibacron blue F3GA. In 50 μl of reaction mixture, 10 μM of the dye could inhibit 50% activity of cinnamomin (7 × 10−9M), whereas 50% inhibition of the enzymatic activity of trichosanthin (7 × 10−9M) and α-sarcin (1 × 10−7M) required 100 and 50 μM of the dye, respectively.

AbstractA new extracellular antifungal protein with a yield of 10 mg per liter was isolated from the culture medium of the mould Trichoderma viride. The protein, which we named viridin, was purified by carboxymethyl-cellulose cation-exchange chromatography and Superose 12 HR 10/30 high-performance liquid chromatography. Viridin, a basic protein of approximately 65 kDa as determined by SDS-PAGE, inhibits the growth of the cotton pathogen Verticillum dahliae, the IC50being 6 ΜM.