The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyltRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.Multiprotein complexes are molecular machines that are essential for organization of the proteome and for integration of cellular functions. Translation of genetic information involves several supramolecular assemblies, including the ribosome and multiprotein complexes involved in the initiation and elongation steps of the protein biosynthesis process (1). A complex containing the four subunits of elongation factor 1 (EF1A and the guanine nucleotide exchange factors EF1B␣, EF1B␤, and EF1B␥) and ValRS (valyl-tRNA synthetase) was described (2, 3). Several structural models of the ValRS-EF1A-guanine nucleotide exchange factor assembly have been proposed (2,4,5). This complex is believed to play a role in channeling of tRNA Val during translation (6). A multiaminoacyl-tRNA synthetase complex (MARS) 4 of about 1.5 MDa was described more than 20 years ago, but its physical and functional organization remain elusive (7). This complex is ubiquitous from Drosophila to mammals and contains the nine aminoacyl-tRNA synthetases ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, ProRS, and the three nonsynthetase components p18, p38, and p43 (8). Initial protein-protein interaction maps have been determined by using the yeast two-hybrid system (9, 10) or by in vitro cross-linking (11). A structural working model has been proposed by electron microscopy and three-dimensional reconstruction (12). The p38 component forms the platform for complex assembly (9, 13). A p38 deficiency is lethal in mice (14). Recent studies have indicated that some of the components of this complex lead a double life. They are essential components of translation when associated within MARS but may also play noncanonical functions after dissociation from the complex or following transport in other cellular compartments. LysRS associates wit...

A dual in vitro/in vivo approach is used to show that WAVE directly binds Ena/VASP, coordinating its activity with that of the Arp2/3 complex for enhanced actin assembly.

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.A wealth of data have suggested that the cytoplasm of human cells is highly organized. This is especially the case as far as translation machinery is concerned. Translation of genetic information involves several supramolecular assemblies, including the ribosome and multiprotein complexes involved in the initiation and elongation steps of the protein biosynthesis process (1). As opposed to their bacterial counterparts, aminoacyl-tRNA synthetases and elongation factors have been described as large molecular machines in human cells. Among the 20 aminoacyl-tRNA synthetases, nine of them (ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, and ProRS) are associated with the three structurally important proteins p18, p38, and p43 to form a multi-aminoacyl-tRNA synthetase (MARS) 4 complex (2-4), and one of them (ValRS) forms the ValRS-EF1A-GEF assembly (VEGA) complex with the four subunits of elongation factor 1 (EF1A, and the guanine nucleotide exchange factors EF1B␣, EF1B␤, and EF1B␥) (5-7).Global analysis of mRNA localization suggests that subcellular localization of proteins occurs at the level of translation, through mRNA localization (8). Colocalization of several components of the translation machinery, including ribosomes, mRNAs, initiation and elongation factors, and aminoacyltRNA synthetases can be easily observed in dendrites of neural cells (9, 10). Association of aminoacyl-tRNA synthetases and elongation factors with the cytoskeletal framework (11-13), and localization of aminoacyl-tRNA synthetases in the vicinity of ribosomes (14) have been described. Several pieces of evidence led to the suggestion that the cytoskeleton may serve as a scaffold to organize components of translation (15, 16). Direct evidence of in vivo compartmentalization of components of translation machinery remains elusive.Recent studies have also indicated that some components of th...

Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis.

For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient’s data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe.

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP’s Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics.

Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNA Met and catalyzes the synthesis of methionyl-tRNA Met . The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA-protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA-protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes.

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.Aminoacyl-tRNA synthetases are essential components of the translation machinery in all living cells. They synthesize aminoacyl-tRNA and therefore establish the genetic code by catalyzing a univocal link between an amino acid and the nucleotide triplet from the anticodon loop of the tRNA molecule (1). Despite the prevalence of this process, tRNA, aminoacyl-tRNA, and aminoacyl-tRNA synthetases are also involved in essential secondary roles (2, 3). It is therefore fundamental to understand the rules that govern their functioning in parallel pathways and to decipher the mechanisms responsible for their spatio-temporal regulation. Recent studies have suggested that the emergence of supramolecular assemblies that serve as depots for releasable regulatory proteins would be a means to control their activities in space and in time (4).A characteristic feature of aminoacyl-tRNA synthetases in animal cells is their ability to form supramolecular complexes. A multi-aminoacyl-tRNA synthetase complex (MARS) 3 containing the nine aminoacyl-tRNA synthetases ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, ProRS, and the three nonsynthetase components p18, p38, and p43 has been extensively characterized (5, 6). It is noteworthy that the complexes isolated from the arthropod Drosophila melanogaster (7), or from various vertebrates, including rat (...