Functional Study Of Ribosome Biogenesis Factor Ltv1 In Zebrafish

The ribosome is found within all living cells.As the site of protein translation,ribosome underpins nearly all activities of cells.Synthesis of ribosomes is one of the most energy-consuming processes in cells.In eukaryotic cells,ribosome biogenesis requires the coordinated activity of all of the three RNA polymerases?RNAP I,II,III?,transcription and maturation of 4 ribosomal RNAs?rRNAs?and approximately 70 small nucleolar RNAs?snoRNAs?,and synthesis of 82 core ribosomal proteins?RPs?and more than 200 non-ribosomal proteins.Impaired ribosome biogenesis and function caused by genetic abnormalities underlie a group of human disorders,collectively known as ribosomopathies.Although all of them involve ribosomal dysfunction,these diseases show different clinical manifestations,mechanisms and potential treatment.Clinical features of ribosomopathies can include defects in digestive organs and craniofacial skeletons,hematological abnormalities and increased risk of neoplastic transformation.Numerous genetic models have been established for the investigation of the mechanisms underlying ribosomopathies.As an ideal model organism in the study of early embryonic development,zebrafish has been more and more used to investigate ribosomopathies.In addition to causative genes in ribosomopathies,some other genes involved in ribosome biogenesis were either mutated or knocked down in zebrafish.These models have revealed new candidate genes of ribosomopathies and are critical resources for the study of the mechanisms underlying ribosomopathies and development of novel therapies.Ltv1 is a non-ribosomal protein that is required for the transport of the 40 s ribosomal subunit.Knockout of ltv1 led to defects in 18 s rRNA processing in yeast,fruit fly and human cells.In addition,cell growth was inhibited in ltv1 loss-of-function yeast strains.The ltv1 deficient fruit fly larvae exhibited development delay and lethality at the second larvae stage.These studies suggest the conserved role of ltv1 in ribosome biogenesis from yeast to multicellular animals.However,the function of ltv1 in development of vertebrates remains poorly understood.In this study,ltv1 was knocked out in zebrafish using CRISPR/Cas9 technology and two mutant alleles,ltv1?7 and ltv1?14,were obtained.18 s rRNA processing was disrupted in the ltv1^-/-mutant,which suggested a conserved role of ltv1 in rRNA processing.At 5 dpf,the ltv1^-/-mutant exhibited aplasia in head,pericardia edema,underdeveloped intestine,smaller liver,uninflated swim bladder and impaired yolk absorption.Ribosomopathy patients often showed defects in digestive organs and craniofacial skeletons and hematological abnormalities,so we analyzed these aspects in the ltv1^-/-mutant.The ltv1^-/-mutant displayed severe abnormalities in craniofacial cartilage,including smaller Meckel's cartilage,curly palatoquadrate and lack of ceratohyal and five ceratobranchial cartilages.The ltv1^-/-mutant showed reduced size of liver,intestine and exocrine pancreas,while the initiation and specification of liver were not impaired.Hematopoietic stem and progenitor cells?HSPCs?,as well as definitive erythrocytes,lymphocytes and myeloid cells were reduced obviously and the primitive hematopoiesis was not affected.These phenotypic features of the ltv1^-/-mutant were reminiscent of the clinical symptoms of some ribosomathies.Further characterization revealed that the defects of exocrine pancreas and HSPCs in ltv1^-/-embryos were due to impaired cell proliferation of ptf1a+ progenitor cells and runx1+ HSPCs,respectively,instead of cell death.It was reported that loss-of-function of genes involved in ribosome biogenesis often caused phenotypes in a P53 dependent manner.In the ltv1^-/-mutant,Although p53 target genes?113p53 and p21 were up regulated,knock down of p53 failed to rescue the developmental abnormalities in ltv1^-/-mutant.Taken together,we generated ltv1^-/-zebrafish mutant and showed the conserved role of ltv1 in 18 s rRNA processing.In addition,ltv1 was shown essential for the development of digestive organs and craniofacial cartilage and hematopoiesis.Further investigation revealed up-regulation of p53 target genes?113p53 and p21,but down-regulation of p53 could not rescue the phenotypes.In short,we generated a new zebrafish model of ribosomopathy,which not only provides a new candidate gene for the screening of ribosomopathies with unkown genetic deterioration,but also serves as a tool to investigate molecular and cellular mechanisms underlying ribosomopathies.