The Mechanism Underlying Prpf31 Deficiency-induced Retinal Development Defect And Retinitis Pigmentosa

Before translation,eukaryotic m RNA needs to undergo splicing process in the nucleus,that is,through the action of spliceosomes to remove introns precisely and link exons,and then mature m RNA is obtained.As an important post-transcriptional regulation mechanism,this process has important biological significance for differential gene expression,developmental regulation of tissues and organs,and maintenance of normal physiological functions.Therefore,abnormal splicing function often leads to severe developmental defects and a variety of human diseases(such as maxillofacial dysplasia,microcranial malformation,RP,aplastic anemia,multiple muscular dystrophy,cancer,etc.).As an important constitutive splicing factor,PRPF31 is widely expressed in various tissues of the organism.Its main function in cells is to promote the assembly and stability of the splicing complex subunit U4/U6.U5 tri-sn RNPs.PRPF31 mutation is one of the main factors causing autosomal dominant retinitis pigmentosa(ad RP).Up to now,more than 130 pathogenic mutation sites of this gene have been reported,but the pathogenic mechanism of this gene mutation causing RP is still unclear.Due to the lack of an ideal animal model,current studies are mainly focused on the cellular level.Therefore,the splicing regulation function of this gene in vivo and the specific mechanism that causes RP and even developmental abnormalities after the deletion are still unclear.PRPF31 is highly conserved evolutionarily,with zebrafish sharing 83% sequence homology with human PRPF31.In addition,zebrafish are very similar to humans in the development,anatomical structure and photoreceptor function of the retina,and have significant advantages as a model animal for ophthalmic diseases.Therefore,in this study,zebrafish was used to investigate the molecular functions,pathogenic mechanisms and functions of Prpf31 in retinal development.In situ hybridization experiments showed that prpf31 maintained a high expression level throughout early retinal development,suggesting that Prpf31 plays an important role in the neurogenesis and function maintenance of the retina.In this study,the prpf31 knockout zebrafish model was successfully constructed by CRISPR/Cas9 technology.Detailed histological and immunofluorescence analyses showed that prpf31 mutation resulted in reduced differentiation of various retinal neurons and Müller glial cells,while retinal progenitor cells(RPCs)showed an "accumulation" state.The overexpression of different types of human PRPF31 in zebrfish showed that the normal m RNA had a better effect on the mutant phenotype,and the p.Lys257fs*277 m RNA had no obvious effect,while that of p.Arg372Glnfs*99 m RNA showed a negative dominant effect.To find out the cytological mechanism of retinal differentiation defect,TUNEL and AO was used to detect a large amount of apoptosis in the mutant retina,and quantitative results detected the activation of p53 pathway.Simultanous deletion of p53 in prpf31 mutants effectively inhibited the occurrence of apoptosis in retinal RPCs,but had no significant effect on differentiated phenotypes,suggesting that p53-mediated apoptosis is more likely to be a consequence than a cause of the failure of RPCs to differentiate.The cell proliferation,mitosis and spindle assembly of the retina were detected by EDU assay,real-time dynamic observation and anti-alpha-tubulin immunofluorescence experiments.It was found that defects in spindle assembly caused RPCs arrested in M phase,and then undergo apoptosis.The DNA integrity was further analyzed by immunofluorescence,comet assay and WB assay,and it was found that prpf31 mutation led to DNA damage in early(36 hpf)retinal cells,accompanied by increased expression of p53 protein.These results suggest that mitotic arrest and apoptosis induced by DNA damage are the cytological basis of retinal differentiation defects.To reveal the molecular mechanism of retinal dysplasia and cytological defects,we used RNA-seq and splicing analysis to find that Prpf31 regulates alternative splicing of a series of important target genes,which are mainly involved in the process of mitosis and DNA damage repair.More importantly,by comparing the splicing differences between the mutant and wild type in different tissues,we found that deletion of Prpf31 had a more significant effect on retinal splicing than in other tissues.This result confirmed the sensitivity of the retina to Prpf31 expression level at the molecular level.Finally,bioinformation analysis and minigene experiments confirmed that the corresponding exon sequences of these abnormal splicing events were short and the possess a weak 5’ splicing site,so they were more dependent on Prpf31.In the absence of this factor,incorrect splicing forms were more likely to be processed.In summary,this study uses the prpf31 knockout zebrafish model to confirm the importance of this gene for the differentiation and survival of RPCs in the process of retinal neurogenesis,and found that Prpf31 is important in the indirect regulation of the splicing of target genes in cells,The changes in these biological processes in the prpf31 mutant are likely to be the pathological mechanisms that cause RP.These results not only clarify the physiological functions of Prpf31 during retinal development,but also provided a reasonable speculation for revealing the molecular mechanism of PRPF31 mutation leading to RP.