Deletion Of Trmu(Mtu1) And Mto1 In Zebrafish Revealed The Essential Role Of TRNA Modification In Mitochondrial Biogenesis And Associated Diseases

During the decoding process of mitochondrial translation,the modification of tRNA wobble position（U34）are crucial to maintain the stability of the corresponding tRNA structure,aminoacylation function,accurate recognition of its codons and to ensure accurate translation of proteins in mitochondria.Several oxidative phosphorylation（OXPHOS）diseases are caused by defects in the post-transcriptional modification of U34in mitochondrial tRNAs.For example,mutations in MTO1 or GTPBP3 impair the 5-taurinomethyluridine（τm⁵U）modification of the wobble uridine and cause hypertrophic myocardium,and mutations in TRMU affect the 2-thiouridine（s²U）modification of U34and cause liver disease.Our lab previous investigations showed that mutations in these three genes（MTO1,GTPBP3,TRMU）involved in U34 modification can modulate the phenotypic manifestation of deafness-associated mitochondrial 12S r RNA mutation.TRMU（also known as MTU1）is a highly conserved tRNA modifying enzyme for the biosynthesis of s²U at the wobble position of tRNA^Gln,tRNA^Glu and tRNA^Lys.However,the pathophysiology of TRMU deficiency remains poorly understood.We generated a trmu knock-out zebrafish mutant by introducing a 32 bp insertion in the exon4 of trmu.And successful trmu mutant were further confirmed by Sanger sequencing,DNA-PAGE and Western blot analyses.We demonstrated the abolished 2-thiouridine modification of U34 of mitochondrial tRNA^Lys,tRNA^Glu and tRNA^Gln in the trmu knock-out zebrafish.The elimination of this post-transcriptional modification mediated mt-tRNA metabolisms,causing the global decreases in the levels of mt-tRNAs.The aberrant mt-tRNA metabolisms led to the impairment of mitochondrial translation,respiratory deficiencies and reductions of mitochondrial ATP production.Compared to wild type embryos,we didn’t find obvious morphological differences in skeletal muscle,brain and retina of trmu^-/-mutant zebrafish at 5 dpf,but its auditory sensory organs,including otoliths and laterline systems,have significantly changed between wildtype and mutant zbrafish,such as the sizes of saccular otolith become smaller and the number of neuromasts in the lateral line system decreases.Strikingly,trmu^-/-mutant zebrafish displayed the abnormal startle response and swimming behaviors.Therefore,our findings may provide new insights into the pathophysiology of deafness,which was manifested by the deficient modifications at wobble position of mt-tRNAs.MTO1 is a highly conserved tRNA modifying enzyme for the biosynthesis ofτm⁵U at the wobble position of mitochondrial tRNA^Glu,tRNA^Gln,tRNA^Lys,tRNA^Trp and tRNA^Leu（UUR）.The previous investigations showed that MTO1 mutations were associated with hypertrophic cardiomyopathy（HCM）.However,the pathophysiology of MTO1deficiency remains elusively and the lack of animal models.Therefore,we got a stable mto1 knock-out zebrafish model by using CRISPR/Cas9 technology,and this model can recapitulate the cardiomyocyte hypertrophy phenotype caused by MTO1 gene mutation in human,such as delayed embryonic heart development and developed hypertrophic cardiomyopathy with the phenotype of enlarged cardiomyocytes.In addition,we found that 2-thiouridine modification and the 5-taurinomethyluridine modification at the wobble position of mt-tRNA are independent.At least the deletion of mto1 didn’t affect the function of trmu.It was also found that deletion of mto1 didn’t affect the steady-state level of mitochondrial tRNA,but it did affect the natural conformation of five mitochondrial tRNA^Gln,tRNA^Glu,tRNA^Lys,tRNA^Trp,tRNA^Leu（UUR）and their aminoacylation levels,which in turn affected the mitochondrial electron transfer chain,expression levels of protein subunits,enzymatic activity of related complexes,and mitochondrial ATP production.Therefore,mitochondrial respiratory dysfunction may be the main cause of hypertrophic cardiomyopathy.