Jiqiang Ling

Aminoacyl-tRNA Synthesis and Translational Quality Control

Translating the 4-letter code of RNA into the 22-letter alphabet of proteins is a central feature of cellular life. The fidelity with which mRNA is translated during protein synthesis is determined by two factors: the availability of aminoacyl-tRNAs composed of cognate amino acid:tRNA pairs and the accurate selection of aminoacyl-tRNAs on the ribosome. The role of aminoacyl-tRNA synthetases in translation is to define the genetic code by accurately pairing cognate tRNAs with their corresponding amino acids. Synthetases achieve the amino acid substrate specificity necessary to keep errors in translation to an acceptable level in two ways: preferential binding of the cognate amino acid and selective editing of near-cognate amino acids. Editing significantly decreases the frequency of errors and is important for translational quality control, and many details of the various editing mechanisms and their effect on different cellular systems are now starting to emerge.

Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site

Oxidative stress arises from excessive reactive oxygen species (ROS) and affects organisms of all three domains of life. Here we present a previously unknown pathway through which ROS may impact faithful protein synthesis. Aminoacyl-tRNA synthetases are key enzymes in the translation of the genetic code; they attach the correct amino acid to each tRNA species and hydrolyze an incorrectly attached amino acid in a process called editing. We show both in vitro and in vivo in Escherichia coli that ROS reduced the overall translational fidelity by impairing the editing activity of threonyl-tRNA synthetase. Hydrogen peroxide oxidized cysteine182 residue critical for editing, leading to Ser-tRNAThr formation and protein mistranslation that impaired growth of Escherichia coli. The presence of major heat shock proteases was required to allow cell growth in medium containing serine and hydrogen peroxide; this suggests that the mistranslated proteins were misfolded.

Post‐transfer editing in vitro and in vivo by the β subunit of phenylalanyl‐tRNA synthetase

Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino‐acid activation and tRNA esterification are corrected by aminoacyl‐tRNA synthetase‐catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino‐acid discrimination is less well understood. We show that phenylalanyl‐tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl‐adenylate and Tyr‐tRNAPhe. Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the β subunit, 40 Å from the active site in the α subunit. Replacements of residues within the editing site had no effect on Phe‐tRNAPhe synthesis, but abolished hydrolysis of Tyr‐tRNAPhein vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded β‐galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino‐acid discrimination in vivo revealed that editing by phenylalanyl‐tRNA synthetase is essential for faithful translation of the genetic code.

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patients clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Childrens Hospital Foundation and others.).

Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNAPhe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNAPhe, an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

Upgrading protein synthesis for synthetic biology

Genetic code expansion for synthesis of proteins containing noncanonical amino acids is a rapidly growing field in synthetic biology. Creating optimal orthogonal translation systems will require re-engineering central components of the protein synthesis machinery on the basis of a solid mechanistic biochemical understanding of the synthetic process.

Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu*GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Mutations in QARS, Encoding Glutaminyl-tRNA Synthetase, Cause Progressive Microcephaly, Cerebral-Cerebellar Atrophy, and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.

Mechanism of tRNA-dependent editing in translational quality control

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNAPhe. Deprotonation of a water molecule by the highly conserved residue βHis-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10-fold. In contrast, substitution of the 3′-OH group of tRNAPhe severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing.

Cell-specific differences in the requirements for translation quality control

Protein synthesis has an overall error rate of approximately 10-4 for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading (“editing”) activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNAPhe. Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the α-subunits contain the active site and the β-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an α-subunit monomer that does not edit Tyr-tRNAPhe, and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNAPhe is synthesized for every ∼7,300 Phe-tRNAPhe, compatible with an error rate in translation of ∼10-4. When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wild-type counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology.