Functional Study Of Isoleucyl-tRNA Synthetase In Zebrafish Digestive Organs Development

Aminoacyl-tRNA synthetases?ARSs?are indispensible for protein synthesis,with specific ARSs catalyzing the ligation of particular amino acids to their cognate tRNAs to ensure the correct transmission of genetic information.In addition to aminoacylation,some ARSs have editing function that prevents incorrect translations when the wrong amino acid binds to a specific codon.Aminoacylation and editing are very typical and conserved functions for ARSs,however,during the evolution from prokaryotes to vertebrates,some ARSs acquired additional domains and thus had some atypical functions:regulation of transcription and translation,signal transduction and cell migration,angiogenesis,inflammatory response and tumorigenesis.More and more studies have shown that the typical and atypical functions of aminoacyl-tRNA synthase have defective,it will lead to human diseases.Aminoacyl-tRNA synthase exists in the cytoplasmic matrix and mitochondria.The aminoacyl-tRNA synthase gene in the cytoplasmic matrix is named ARS,and the aminoacyl-tRNA synthase gene in the mitochondria is named ARS2.Some clinical symptoms caused by the mutation of isoleucyl-tRNA synthetase?IARS?gene in humans have been reported,such as growth retardation,intellectual disability,muscular hypotonia,and infantile hepatopathy.Three of non-related IARS mutants were studied,and the IARS genes were mutated in different ways,with different phenotypic severity.In human individuals with IARS mutations,one allele of IARS is completely loss of function,while the other allele remains partial functions.The phenotypic severity is vary maybe depend on whether the mutation site is located in the conserved domain.Mutations in IARS can cause liver dysfunction,but how IARS mutations affect the development or function of the liver or other digestive organs has not been clearly reported.we got a mutant with severe liver and intestine defects by ENU screen,we carried out positional cloning and found a site mutation in iars.Genomic DNA sequencing of iars gene reveals that a splicing site between exon22 and intron22 is disrupted by a T to A transversion,so the iars^V9/V9mutants lossed exon22,it has 78 bases.The altered transcript is predicted to encode a slightly shorter protein than wild-type,patially loss the anticondon domain,maybe most of protein domains are still present.The mutation of human IARS will cause a series of multi-system diseases.These individuals have common clinical symptoms,such as growth retardation,intellectual disability,muscular hypotonia,and infantile hepatopathy.In order to explore the effects of different phenotypes caused by different mutanted forms of iars on the digestive organs of zebrafish,we used gene editing technology CRISPR/Cas9 to knock out iars in zebrafish and obtained another mutant,iars^+1/+1.The exon7 of the mutant inserted a base,leads to the shift code mutation.It is predicted that iars^+1/+1 mutants encoded only 269 amino acids,while the wild type iars encoded 1271 amino acids,maybe iars^+1/+1 mutants lossed most protein domains.We directly observed the two kinds of mutants under the microscope.At 2dpf,there was no obvious difference between the iars^V9/V9 mutants and the wild type,while the iars^+1/+1mutants are distinguished from the sibling,showed small head and eyes.At 5dpf,iars^V9/V9mutants showed small head and eyes,which is more obvious compared to sibling,showed no swim bladder,pericardial edema and malabsorption of yolk,these phenotypes are more obvious in iars^+1/+1 mutants,and it is easy to distinguish iars^+1/+1 mutants from sibling or iars^V9/V9mutants.According to the death statistics of the two mutants,the survival time of iars^V9/V9mutants was slightly longer than iars^+1/+1 mutants.The iars^V9/V9mutants died from 9dpf to 11dpf,and the iars^+1/+1 mutants died from 6dpf to 9dpf.Compared with iars^V9/V9mutants,iars^+1/+1 mutants showed more severe phenotypes in the digestive organs,craniofacial skeletal,decreased proliferation of liver cells and ER stress.Taken together,our study showed that iars is essential for the development of the digestive organs in zebrafish,and further revealed that the iars^V9/V9mutant was milder than the iars⁺1/+11/+1 mutant,which was consistent with the different phenotypic severity due to different mutation sites in human IARS mutants.