Mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are essential components of the mitochondrial translation machinery. The correlation of mitochondrial disorders with mutations in these enzymes has ...raised the interest of the scientific community over the past several years. Most surprising has been the wide-ranging presentation of clinical manifestations in patients with mt-aaRS mutations, despite the enzymes' common biochemical role. Even among cases where a common physiological system is affected, phenotypes, severity, and age of onset varies depending on which mt-aaRS is mutated. Here, we review work done thus far and propose a categorization of diseases based on tissue specificity that highlights emerging patterns. We further discuss multiple in vitro and in cellulo efforts to characterize the behavior of WT and mutant mt-aaRSs that have shaped hypotheses about the molecular causes of these pathologies. Much remains to do in order to complete our understanding of these proteins. We expect that futher work is likely to result in the discovery of new roles for the mt-aaRSs in addition to their fundamental function in mitochondrial translation, informing the development of treatment strategies and diagnoses.
Metazoan complexity and life-style depend on the bioenergetic potential of mitochondria. However, higher aerobic activity and genetic drift impose strong mutation pressure and risk of irreversible ...fitness decline in mitochondrial (mt)DNA-encoded genes. Bilaterian mitochondria-encoded tRNA genes, key players in mitochondrial activity, have accumulated mutations at significantly higher rates than their cytoplasmic counterparts, resulting in foreshortened and fragile structures. Here we show that fragility of mt tRNAs coincided with the evolution of bilaterian animals. We demonstrate that bilaterians compensated for this reduced structural complexity in mt tRNAs by sequence-independent induced-fit adaption to the cognate mitochondrial aminoacyl-tRNA synthetase (aaRS). Structural readout by nuclear-encoded aaRS partners relaxed the sequence constraints on mt tRNAs and facilitated accommodation of functionally disruptive mutational insults by cis-acting epistatic compensations. Our results thus suggest that mutational freedom in mt tRNA genes is an adaptation to increased mutation pressure that was associated with the evolution of animal complexity.
Abstract
Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of ...these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
The COVID-19 pandemic has challenged undergraduate instructors and students in an unprecedented manner. Each has needed to find creative ways to continue the engaged teaching and learning process in ...an environment defined by physical separation and emotional anxiety and uncertainty. As a potential tool to meet this challenge, we developed a set of curricular materials that combined our respective life science teaching interests with the real-time scientific problem of the COVID-19 pandemic in progress. Discrete modules were designed that are engaging to students, implement active learning-based coursework in a variety of institutional and learning settings, and can be used either in person or remotely. The resulting interdisciplinary curriculum, dubbed "COVID-360," enables instructors to select from a menu of curricular options that best fit their course content, desired activities, and mode of class delivery. Here we describe how we devised the COVID-360 curriculum and how it represents our efforts to creatively and effectively respond to the instructional needs of diverse students in the face of an ongoing instructional crisis.
The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. ...The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.
In all eukaryotic cells, protein synthesis occurs not only in the cytosol, but also in the mitochondria. Translation of mitochondrial genes requires a set of aminoacyl-tRNA synthetases, many of which ...are often specialized for organellar function. These enzymes have evolved unique mechanisms for tRNA recognition and for ensuring fidelity of translation. Mutations of human mitochondrial synthetases are associated with a wide range of pathogenic phenotypes, both highlighting the importance of their role in maintaining the cellular "powerhouse" and suggesting additional cellular roles.
Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino ...acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.
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