DEAD-box Helicase Ddx27 And Ddx19 Deletion Lead To Abnormal Morphology Of Zebrafish

Dead-box RNA helicases are highly conserved in organisms ranging from bacteria to mammals.These enzymes play important roles in pre-m RNA splicing,transcription,ribosomal generation,RNA transport,translation,RNA decay,and the regulation of organelle gene expression.Through these function,the helicases affect body development,immunity and tumorigenesis.At present,the redundancy and specificity of many helicase proteins as well as their specific roles and mechanisms in embryonic development or diseases are still unclear.Here,examined Ddx27 and Ddx19,two members of dead-box helicase family,for their functions in development.Ddx27 is known to be involved in ribosomal biogenesis during RNA metabolism processes.Ddx19 has been linked to nuclear and cytoplasmic transport.They are both highly expressed in tumor cells.Both of them have been implicated in early embryonic development,but their specific functions remain to be defined.Cell apoptosis and cell proliferation are critical to mormal development and functional differentiation of organisms,as these two cellular prcesses manifest growth and development.Apoptosis,or programmed cell death regulated by genes,is a physiological processes closely related to cancer,autoimmunity and degenerative diseases.This process is initiated and regulated by the Caspase family,which comprises seven proteins in zebrafish.The apoptosis signaling pathway is triggered by caspase activation through a variety of independent pathways.Pathways known to mediate apoptosis include the internal mitochondrial pathway,death receptor-mediated external pathway,and endoplasmic reticulum pathway.Cell proliferation is often regulated by the cell cycle.As a conserved mechanism of eukaryotic cell self-replication,cell cycle regulation is crucial for tissue homeostasis and cell replacement during normal organism development.Multiple regulatory factors are involved in the cell cycle regulation process,and any wrong expression will lead to malignant proliferation and differentiation,associated with a variety of diseases and tumors.We speculate that helicases Ddx27 and Ddx19 regulate process of cell proliferation and apoptosis.CRISPR/Cas9 gene editing technology has been deeply applied in basic science research,especially in the research using zebrafish as the experimental model.The model organism is characterized with in vitro fertilization,transparent embryo and strong fecundity,which largely reduce the difficulty level and cost of mutation system preparation.and further facilitate the application of CRISPR/Cas9 gene editing technology.Moreover,zebrafish is an ideal model organism for studies of early embryonic development.Its skeletal muscle system is highly conserved and finely regulated by a variety of signaling pathways.Many of the mutations have phenotypes similar to those seen in human diseases.Therefore,zebrafish has been widely used for studies on vertebrate growth,repair and musculoskeletal system disease.This model organism provides an efficient platform to decipher the pathology and physiology og diseases,and to conduct drug screening.This approach will certainly contribute to the understanding of many major diseases mechanisms and and the search for therapeutic treatment.We used bioinformatics methods to identify the members of DEAD-box family in15 species.The results showed that the family existed from creatures with a low evolutionary status to organisms with a high evolutionary status,and the number of the family members in invertebrates was about 30.As the degree of biological evolution increases,so does the number of members,and the number of members in vertebrates was stable at about 40.The protein structure,physicochemical properties and subcellular localization of each member of the zebrafish DEAD-box family were comprehensively and systematically analyzed.The result shows that the proteins sequences of this family contain 15 conserved motifs,containing D-E-A-D conserved amino acid sequences except ddx47 and ddx58,and they are widely distributed on chromosomes.The relative molecular weight of the protein was larger than 40k D except Ddx41 and Ddx47.In this family,there are 7 stable proteins and 33 unstable proteins,except for Ddx41,others exhibit hydrophobic properties.The protein subcellular localization shows that the major proteins are mainly distributed in the nucleus,followed by the cytoplasm and mitochondria,with a small amount in the plasma membrane,extracellular matrix,endoplasmic reticulum,golgiosome and peroxisomes.According to cluster analysis,the DEAD-box family of zebrafish has a good correspondence with each member in human and mouse,and the evolutionary relationship between different members is close.The support rate of ddx5 and ddx17,eif4a and eif4a3,ddx6 and ddx61 are 100%.This study not only comprehensively and systematically analyzed the information of members in the DEAD-box family of zebrafish,but also analyzed the evolutionary relationship of this family,providing some theoretical guidance for us to deeply understand the family and study the function of the DEAD-box family in zebrafish.To further study the function and mechanism of Ddx27 and Ddx19 in developmental regulation,we obtained ddx27 and ddx19 heritable effective mutants by CRISPR/Cas9 genome editing technology.(1)Ddx27 amino acid sequence is highly conserved,which was up to 87%consistent with human and mouse,and is expressed in early zebrafish embryos continuously.(2)Ddx27 contains 21 exons.We designed the knockout target on the sixth exon,knockout of ddx27 gene by the CRISPR/Cas9 gene editing technology.A total of four effective mutation types were obtained,which were+6 bp,-27 bp,-14 bp and-5 bp,respectively.(3)After morphological observation,we found that the absence of ddx27 showed a phenotype of small head,small eyes,pericardial edema,thin trunk and disappeared pectoral fin and homozygous death.The maximum survival time was no more than 7dpf.(4)Using Alcian blue staining,we found that compared with its sibling,ddx27homozygous cranial cartilage development defects,7 pairs of pharyngeal arch cartilage disappeared.(5)Through Tg(mylpfa:EGFP)transgenic fish line observation,similar to craniofacial cartilage defect phenotype,ddx27 homozygous craniofacial skeletal muscle development was severely impaired.(6)Further detection of cell apoptosis and proliferation by Tunel staining and Edu labeling revealed that the apoptosis signal was significantly up-regulated and the number of cell proliferation was significantly reduced in the ddx27 homozygous mutant compared to sibling.(7)Based on quantitative analysis by q-RT PCR,we found that the expressions of caspase2,caspase3a,caspase3b,caspase6 and bcl2b were significantly down-regulated in ddx27 homozygotes compared to wild-type zebrafish.The transcription levels of tp53was also decreased,while the transcription levels of caspase8,atf4a and atf4b were increased compared with the wild-type at 72 hpf(8)The ddx19 gene is also continuously expressed in early zebrafish embryos,which contains 12 exons,which contains 12 exons and the knockout target is located on the first exon.Two different types of deletion mutations F₁ were obtained by CRISPR/Cas9 gene editing,which were-46 bp and-7 bp respectively.(9)After morphological observation,we found that the ddx19 homozygous mutant showed small head,small eyes,pericardial edema and trunk curvature,which was homozygous to death before 5 dpf.(10)The Tunel staining test showed that apoptosis levels were up-regulated in ddx19 homozygous mutants.Edu labeling detection revealed that,compared with their sibling,the ddx19 delection resulted in a significant decrease in the number of cells in the S phase,and the cell proliferation was largely blocked.In summary,through morphological observation and statistical analysis,we found that ddx27 and ddx19 homozygous mutants were both lethal.ddx27 deletion resulted in a phenotype of small head,small eyes,pericardial edema,trunk thinness and pectoral fin disappearance.ddx19 also caused severe deformity,manifested as,including small head,small eyes,pericardial edema and trunk curvature.Furthermore,using Alcian blue staining and green fluorescent fish line,we found that the craniofacial cartilage and skeletal muscle development of zebrafish ddx27 homozygous mutant were seriously deficient.The Tunel staining test showed that apoptosis levels were up-regulated in ddx27 and ddx19 homozygous mutants.At the same time,Edu labeling detection revealed that,compared with their sibling,the ddx27 or ddx19 delection resulted in a significant decrease in the number of cells in the s phase,and the cell proliferation was largely blocked.In summary,these results suggest that ddx27 affects the formation of cartilage and muscle system by regulating cell apoptosis and proliferation in early embryonic development of zebrafish.Ddx19 is also involved in the regulation of cell apoptosis and proliferation,thus affecting the normal development process.