Identificación y caracterización de nuevos genes implicados en la función OXPHOS y su posible asociación a patologías humanas

Abstract

Abstract 3 Mitochondria are essential organelles for numerous cellular processes, highlighting their role in the production of energy supply in the form of ATP in a process known as oxidative phosphorylation (OXPHOS). The OXPHOS system deficiencies lead to mitochondrial diseases. Mitochondria possess their own genome (mtDNA) which in mammals encodes only 13 proteins, the remaining ̴1500 proteins that comprise the mammalian proteome are encoded by nDNA. At present, there are both a significant number of uncharacterized mitochondrial proteins and mitochondrial diseases in which the affected gene is unknown, making it difficult to diagnose patients and to comprehend these disease mechanisms. Our previous studies revealed that highly conserved essential genes for the OXPHOS function (mt-Tfb1, GatC, Pol- and Coa3) are encoded by bicistronic mRNAs in Drosophila, suggesting a tendency for mitochondrial genes to be organized in these particular mRNAs in this organism. Thereby, it could be employed as a tool for identifying evolutionary conserved non-described genes. This thesis reinforces the previous observation, identifying a new and previously undescribed gene involved in OXPHOS function, as well as describes a novel mutation in a human orthologue of a gene encoded by a bicistronic mRNA in Drosophila. Thus, we characterized the first pathogenic mutation in the GatC subunit of the heterotrimeric enzyme glutamyl-tRNAGln amidotransferase (GatCAB) in a patient presented with severe cardiomyopathy and lactic acidosis. GatCAB participates in the synthesis of Gln-tRNAGln in response to the absence of a mitochondrial glutaminyl-tRNA synthetase (QARS2). Patient-derived fibroblasts showed that a reduction in the steady-state levels of GATC leads to a decrease in the rest of the subunits that constitute the enzyme, GATA and GATB. We also demonstrate that a reduction of these subunits causes a severe mitochondrial protein translation system defect, corroborating the functional role of the transamidation route in human mitochondria. On the other hand, the analysis of candidate genes to participate in the OXPHOS function encoded in biscitronic mRNAS in Drosophila, allowed us to identify and partially characterized C6orf203, a 19 kDa protein localized in the mitochondrial matrix. Its functional characterization using the genomic editing CRISPR/Cas9 system allowed us to conclude that C6orf203 participates in the mitochondrial translation system and forms high-molecular-weight complexes above 1000 kDa. In conclusion, during this study we have made progress both in the characterization of a gene linked, for the first time, to human mitochondrial pathology and in the identification and characterization of a novel protein involved in the mitochondrial OXPHOS function.