La Traducció genètica mitocondrial i malalties associades

En humans, com en la majoria d'organismes eucariotes, la síntesi proteica té lloc simultàniament al citoplasma i en orgànuls que posseeixen un genoma propi. Els mitocondris requereixen una maquinària traduccional pròpia per sintetitzar els tretze polipèptids, codificats al genoma mitocondrial, que formen part dels complexos de la cadena respiratòria i la fosforilació oxidativa responsables de proporcionar energia a la cèl·lula. Els elements que componen aquesta maquinària es troben codificats tant al genoma mitocondrial com al nuclear i participen de manera coordinada en la traducció genètica. Mutacions en els gens que codifiquen aquests factors de l'aparell de traducció genètica mitocondrial desencadenen un ampli ventall de malalties greus en humans, caracteritzades per símptomes heterogenis que en dificulten el diagnòstic i tractament. Hi ha malalties mitocondrials humanes causades per mutacions en el DNA mitocondrial que afecten específicament els tRNA i rRNA i, a més, s'han descrit mutacions en proteïnes mitocondrials codificades en el genoma nuclear, entre les quals es troben mutacions en factors de traducció, enzims de processament i modificació dels tRNA, proteïnes mitoribosòmiques i aminoacil-tRNA-sintetases mitocondrials. La complexitat de les malalties mitocondrials, la varietat de símptomes que causen i la dificultat de manipular genèticament el DNA mitocondrial compliquen la recerca relacionada amb aquestes malalties i justifiquen la generació de models animals que permetin caracteritzar-les i desenvolupar noves estratègies terapèutiques.In humans, as in the majority of eukaryotic organisms, protein synthesis occurs simultaneously in the cytoplasm and in those organelles that possess their own genome. Mitochondria require its own translational machinery in order to synthesize the 13 polypeptides, encoded in the mitochondrial genome, which are part of the respiratory chain and the oxidative phosphorylation complexes, responsible for supplying energy to the cell. The elements that compose this machinery are encoded both in the mitochondrial and the nuclear genome, and participate in gene translation in a coordinate manner. Mutations in genes that code for these factors of the gene translation apparatus trigger a wide range of severe pathologies in humans, characterized by heterogeneous symptoms that difficult their diagnostic and treatment. There exist human mitochondrial diseases caused by mutations in the mitochondrial DNA which specifically affect tRNA and rRNA and, additionally, mutations in nuclear encoded mitochondrial proteins have been described, among which are mutations in translation factors, enzymes involved in tRNA processing and modification, mitoribosomal proteins, and aminoacyl-tRNA synthetases. The complexity of mitochondrial pathologies, the variety of symptoms they cause, and the difficulty to manipulate mitochondrial DNA complicate the research related to these diseases and justify the generation of animal models that allow their characterization and the development of new therapeutic strategies

Aminoacyl-tRNA synthetases: a complex system beyond protein synthesis

Les aminoacil-tRNA sintetases (ARSs) són els enzims que tradueixen el codi genètic unint aminoàcids a l'RNA de transferència (ARNt) corresponent. Els tRNA aminoacilats poden ser utilitzats aleshores pel ribosoma per traduir RNA missatgers (mRNA). El rol essencial de les ARS es va establir en la dècada dels seixanta, durant l'era d'or de la biologia molecular, que va dur al descobriment del codi genètic. El paper canònic d'aquests enzims es troba actualment descrit en tots els llibres de text. Tot i això, l'interès per la funció de les ARS continua creixent extraordinàriament, a causa de les noves i inesperades funcions descobertes per a aquests enzims, per a l'ARNt i per a l'ARN en general. En aquest article descriurem els darrers progressos en l'estudi de les aminoacil-tRNA sintetases, resumirem els coneixements actuals sobre l'evolució de les ARS, introduirem els lectors en diverses facetes de la biologia cel·lular en què s'ha comprovat que les ARS tenen un paper important i discutirem les aplicacions derivades d'aquests estudis.Aminoacyl-tRNA synthetases (ARSs) are enzymes that translate the genetic code by adding amino acids to their cognate transfer RNAs (tRNA). Aminoacylated tRNAs can then be used by the ribosome to decode mRNA. The essential role of ARSs was established in the 1960s, during the golden era of molecular biology that led to the discovery of the genetic code. The canonical role of these enzymes is now described in all textbooks. Remarkably, however, interest in ARS function continues to grow as new and unexpected functions are discovered for these enzymes, for tRNA, and for RNA in general. This article describes current progress in the field of ARS research, summarizes current thinking about the evolution of ARSs, introduces the readers to the many facets of cellular biology in which ARSs play an important role, and discusses the biotechnological applications derived from these studies

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family

Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base “mutations” when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research

Chimeric tRNAs as tools to induce proteome damage and identify components of stress responses

Misfolded proteins are caused by genomic mutations, aberrant splicing events, translation errors or environmental factors. The accumulation of misfolded proteins is a phenomenon connected to several human disorders, and is managed by stress responses specific to the cellular compartments being affected. In wild-type cells these mechanisms of stress response can be experimentally induced by expressing recombinant misfolded proteins or by incubating cells with large concentrations of amino acid analogues. Here, we report a novel approach for the induction of stress responses to protein aggregation. Our method is based on engineered transfer RNAs that can be expressed in cells or tissues, where they actively integrate in the translation machinery causing general proteome substitutions. This strategy allows for the introduction of mutations of increasing severity randomly in the proteome, without exposing cells to unnatural compounds. Here, we show that this approach can be used for the differential activation of the stress response in the Endoplasmic Reticulum (ER). As an example of the applications of this method, we have applied it to the identification of human microRNAs activated or repressed during unfolded protein stress

Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation

Mitochondria are subcellular organelles critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, as well as their coordinated translation, import and respiratory complex assembly. Here we describe the characterization of exonuclease domain like 2 (EXD2), a nuclear encoded gene that we show is targeted to the mitochondria and prevents the aberrant association of mRNAs with the mitochondrial ribosome. The loss of EXD2 resulted in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of EXD2/CG6744 in D.melanogaster caused developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that could be reversed with an anti-oxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and aging