Magali Frugier

Pathogenic variants in mitochondrial aminoacyl-tRNA synthetases result in a broad range of mitochondrial respiratory chain disorders despite their shared role in mitochondrial protein synthesis. LARS2 encodes the mitochondrial leucyl-tRNA synthetase, which attaches leucine to its cognate tRNA. Sequence variants in LARS2 have previously been associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss (OMIM #615300). In this study, we report variants in LARS2 that are associated with a severe multisystem metabolic disorder. The proband was born prematurely with severe lactic acidosis, hydrops, and sideroblastic anemia. She had multisystem complications with hyaline membrane disease, impaired cardiac function, a coagulopathy, pulmonary hypertension, and progressive renal disease and succumbed at 5 days of age. Whole exome sequencing of patient DNA revealed compound heterozygous variants in LARS2 (c.1289C>T; p.Ala430Val and c.1565C>A; p.Thr522Asn). The c.1565C>A (p.Thr522Asn) LARS2 variant has previously been associated with Perrault syndrome and both identified variants are predicted to be damaging (SIFT, PolyPhen). Muscle and liver samples from the proband did not display marked mitochondrial respiratory chain enzyme deficiency. Immunoblotting of patient muscle and liver showed LARS2 levels were reduced in liver and complex I protein levels were reduced in patient muscle and liver. Aminoacylation assays revealed p.Ala430Val LARS2 had an 18-fold loss of catalytic efficiency and p.Thr522Asn a 9-fold loss compared to wild-type LARS2. We suggest that the identified LARS2 variants are responsible for the severe multisystem clinical phenotype seen in this baby and that mutations in LARS2 can result in variable phenotypes.

RNA recognition by tRNA synthetases is thought to have arisen by the recruitment of RNA binding peptide elements into the frameworks of primordial enzymes that carried out amino acid activation. While peptides have been shown to have the capacity to discriminate irregular RNA structures such as bulges and loops, the sequence-specific recognition of base pairs in RNA helices like those in tRNAs had not been demonstrated. Such discrimination by peptide binders was thought to be inherently difficult. But in this work we show that discrimination of a single base pair in an RNA helix may be achieved with a rationally designed, chemically synthesized peptide. In a separate study, we demonstrated the noncovalent association of a peptide binder with an amino acid activation domain to give an active and specific tRNA synthetase. Thus, peptides with high specificity for an RNA helix can be obtained. In addition, a peptide can associate with another protein to give an enzyme that acts on RNA. The principles suggested by these studies may be general.

The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but two mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5'-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.

We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contempo...

This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation. The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species. Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon.

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA(Asn). However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA(Asn) accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA(Asn) in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as "tRNA sponges" and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.

As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.

Distinctive features of aspartyl-transfer RNA (tRNA) synthetases (AspRS) from the protozoan Plasmodium genus are described. These apicomplexan AspRSs contain 29-31 amino acid insertions in their anticodon binding domains, a remarkably long N-terminal appendix that varies in size from 110 to 165 amino acids and two potential initiation codons. This article focuses on the atypical functional and structural properties of Plasmodium falciparum cytosolic AspRS, the causative parasite of human malaria. This species encodes a 626 or 577 amino acids AspRS depending on whether initiation starts on the first or second in-frame initiation codon. The longer protein has poor solubility and a propensity to aggregate. Production of the short version was favored as shown by the comparison of the recombinant protein with endogenous AspRS. Comparison of the tRNA aminoacylation activity of wild-type and mutant parasite AspRSs with those of yeast and human AspRSs revealed unique properties. The N-terminal extension contains a motif that provides unexpectedly strong RNA binding to plasmodial AspRS. Furthermore, experiments demonstrated the requirement of the plasmodial insertion for AspRS dimerization and, therefore, tRNA aminoacylation and other putative functions. Implications for the parasite biology are proposed. These data provide a robust background for unraveling the precise functional properties of the parasite AspRS and for developing novel lead compounds against malaria, targeting its idiosyncratic domains.

Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.

Structural plasticity of transfer RNA (tRNA) molecules is essential for interactions with their biological partners in aminoacylation reactions and during ribosome-dependent protein synthesis. This holds true when tRNAs are recruited for other functions than translation. Here we review regulation pathways where tRNAs and tRNA mimics play a pivotal role. We further discuss the importance of the identity signals used in aminoacylation that are also required to specify regulatory mechanisms. Such mechanisms are diverse and intervene in transcription, splicing and translation. Altogether, the review highlights the many manners architectural features of tRNA were selected by evolution to control biological key processes.

We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity. A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine. The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop. The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate. The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.