Konckout Of Mitochondrial TRNA Nuclear-modifier Genes Gtpbp3,mto1 And Trmu Mediated By Cas9 In Zebrafish

GTPBP3, MTO1 and TRMU are proteins that are encoded by nuclear DNA(nDNA) and are subsequently imported into the mitochondrion to function. They take part in the process of 5-taurinomethyl-2-thiouridine (Tm5S2U) modification of mitochondrial tRNAs(mt-tRNAs) at U34 site. Among them, GTPBP3 and MTO1 are responsible for the biosynthesis of 5-taurinomethyluridine (τm5U) in mt-tRNA Leu(UUR) and tRNATrp by forming a heterotetrameric α2β2 complex. TRMU together with GTPBP3 and MTO1 are involved in the biosynthesis of the τm5S2U through a distinct pathway.In human, it has been reported that mutations of GTPBP3, MTO1 and TRMU are associated with human mitochondrial disease. Mutations in TRMU can lead to acute infantile liver failure; mutations in MTO1 are found to underlie cases of hypertrophic cardiomyopathy and lactic acidosis; and more recently, mutations in GTPBP3 cause a mitochondrial translation defect associated with hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy.Recently, the study in human cell indicated that MTO1 and TRMU may have other cellular functions apart from mt-tRNAs modification. MTO1 has a new and surprising role in interacting with mitoribosomal subunits and affecting mitoribosome assembly; TRMU might be involved in the assembly of enzyme complexes containing iron-sulfur clusters(e.g., Complex II of the respiratory chain).To further decipher the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of mitochondrial disease due to mutations in these 3 genes. We conduct the bioinformatics analysis and spatio-temporal expression pattern analysis of gtpbp3, mtol and trmu. Further more, we construct the zebrafish mutants of 3 genes using CRISPR/Cas9 techniques.Homologous alignment shows that the amino acid sequence of 3 proteins are relatively conservative among the diverse species and the sequence identity between zebrafish and human of three proteins are all more than 60%. Some important domains of these proteins are highly conservative too.The result of qRT-PCR shows that gtpbp3, mtol and trmu are ubiquitously expressed in various tissues of adult zebrafish, but extremely abundant in ovary; and relatively high in tissues with high metabolic rates including eye, heart, and kidney. We further determine that the mRNA and protein level of gtpbp3 is high in unfertilized eggs and it is expressed in all stages of zebrafish early embryonic development. These results demonstrate that 3 genes may be crucial for early embryonic development and are relatively more important to the tissues that have high ATP demand.Finally, using the technology CRISPR/Cas9, we gain the gtpbp3△14bp and gtpbp3△13bp zebrafish mutants of gtpbp3; and the mtol△7bp and mto1△8bp mutants of mtol in zebrafish; and the trmu+32bp mutants of trmu gene. Except the gtpbp3△14bp, all others mutations can induce gene disruption including frameshift mutation and nonsense mutation.Hence, we successfully construct the zebrafish mutants of gtpbp3, mtol and trmu. This will be in favour of gaining further understanding of the fuction of these genes and the precise pathogenic mechanism of mitochondrial disease due to mutations in these 3 genes.