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FARS2 deficiency in Drosophila reveals the developmental delay and seizure manifested by aberrant mitochondrial tRNA metabolism.

ABSTRACT: Mutations in genes encoding mitochondrial aminoacyl-tRNA synthetases are linked to diverse diseases. However, the precise mechanisms by which these mutations affect mitochondrial function and disease development are not fully understood. Here, we develop a Drosophila model to study the function of dFARS2, the Drosophila homologue of the mitochondrial phenylalanyl-tRNA synthetase, and further characterize human disease-associated FARS2 variants. Inactivation of dFARS2 in Drosophila leads to developmental delay and seizure. Biochemical studies reveal that dFARS2 is required for mitochondrial tRNA aminoacylation, mitochondrial protein stability, and assembly and enzyme activities of OXPHOS complexes. Interestingly, by modeling FARS2 mutations associated with human disease in Drosophila, we provide evidence that expression of two human FARS2 variants, p.G309S and p.D142Y, induces seizure behaviors and locomotion defects, respectively. Together, our results not only show the relationship between dysfunction of mitochondrial aminoacylation system and pathologies, but also illustrate the application of Drosophila model for functional analysis of human disease-causing variants.

SUBMITTER: Fan W

PROVIDER: S-EPMC8682739 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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FARS2 deficiency in Drosophila reveals the developmental delay and seizure manifested by aberrant mitochondrial tRNA metabolism.

Fan Wenlu W Jin Xiaoye X Xu Man M Xi Yongmei Y Lu Weiguo W Yang Xiaohang X Guan Min-Xin MX Ge Wanzhong W

Nucleic acids research 20211201 22

Mutations in genes encoding mitochondrial aminoacyl-tRNA synthetases are linked to diverse diseases. However, the precise mechanisms by which these mutations affect mitochondrial function and disease development are not fully understood. Here, we develop a Drosophila model to study the function of dFARS2, the Drosophila homologue of the mitochondrial phenylalanyl-tRNA synthetase, and further characterize human disease-associated FARS2 variants. Inactivation of dFARS2 in Drosophila leads to devel ...[more]

PMID: 34878141

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Project description:Recent developments in molecular cytogenetics allow the detection of genomic rearrangements at an unprecedented level leading to discoveries of previously unknown chromosomal imbalances (zygotic and post-zygotic/mosaic). These can be accompanied by a different kind of pathological genome variations, i.e. chromosome instability (CIN) manifested as structural chromosomal rearrangements and low-level mosaic aneuploidy. Fortunately, combining whole-genome and single-cell molecular cytogenetic techniques with bioinformatics offers an opportunity to link genomic changes to specific molecular or cellular pathology. High-resolution chromosomal SNP microarray analysis was performed to study the genome of a 15-month-aged boy presented with developmental delay, congenital malformations, feeding problems, deafness, epileptiform activity, and eye pathology. In addition, somatic chromosomal mutations (CIN) were analyzed by fluorescence in situ hybridization (FISH). Interstitial 5p13.3p13.2 duplication was revealed in the index patient. Moreover, CIN manifested almost exclusively as chromosome losses and gains (aneuploidy) was detected. Using bioinformatic analysis of SNP array data and FISH results, CIN association with the genomic imbalance resulted from the duplication was proposed. The duplication was demonstrated to encompass genes implicated in cell cycle, programmed cell death, chromosome segregation and genome stability maintenance pathways as shown by an interactomic analysis. Genotype-phenotype correlations were observed, as well. To the best our knowledge, identical duplications have not been reported in the available literature. Apart from genotype-phenotype correlations, it was possible to propose a link between the duplication and CIN (aneuploidy). This case study demonstrates that combining SNP array genomic analysis, bioinformatics and molecular cytogenetic evaluation of somatic genome variations is able to provide a view on cellular and molecular pathology in a personalized manner. Therefore, one can speculate that similar approaches targeting both interindividual and intercellular genomic variations could be useful for a better understanding of disease mechanisms and disease-related biological processes.

Project description:Background & aimsCongenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.MethodsWe collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.ResultsIn the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.ConclusionsWe identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

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FARS2 deficiency in Drosophila reveals the developmental delay and seizure manifested by aberrant mitochondrial tRNA metabolism.

Publications

FARS2 deficiency in Drosophila reveals the developmental delay and seizure manifested by aberrant mitochondrial tRNA metabolism.

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