| The spliceosome is a molecular machine that functions in pre-m RNA splicing.More than 95% of eukaryotic genes undergo pre-m RNA splicing,to remove introns and ligate exons,by which processed into mature m RNA to guide protein synthesis.Pre-m RNA splicing is a highly complicated and exquisitely regulated process,which is essential for the phylogeny and maintenance of tissue homeostasis.Defects in pre-m RNA splicing often lead to human diseases,which affecting tissues as diverse as retina,hematopoietic lineage,craniofacial skeleton,spinal cord,and limbs,etc.Splicing factors have been shown to play a crucial role in the development of hematopoietic system,but the specific regulatory mechanisms remain unclear.Pre-m RNA processing factor 31(PRPF31)is a U4 small nuclear ribonucleoproteins(sn RNPs).As a splicing factor,PRPF31 participates in prem RNA splicing process by promoting the assembly and stability of U4/U6·U5 Tri-sn RNPs.Mutations in human PRPF31 gene cause autosomal dominant retinitis pigmentosa,however PRPF31 has not yet been reported to participate in the development of hematopoietic system.Zebrafish,as a classical animal model,has been widely used in the study of hematopoietic system development.In this study,zebrafish was ultilized as a model to explore the function and regulating mechanism of zebrafish prpf31 gene in the development of hematopoietic system.In situ hybridization was used to examine the expression of prpf31 in zebrafish during early embryogenesis.The results showed that prpf31 was significantly expressed in a series of primitive hematopoietic,definitive hematopoietic,and vascular regions during early embryogenesis,suggesting that Prpf31 may be involved in the development of hematopoietic and vascular system.The role of Prpf31 in the development of zebrafish hematopoiesis was observed in prpf31 knockout zebrafish by in situ hybridization.The results showed that prpf31 knockout zebrafish exhibited normal primary hematopoiesis and erythromyeloid progenitor hematopoiesis.However,during the definitive hematopoietic process,hematopoietic stem and progenitor cells(HSPCs)and its differentiated multiple blood lineages were severely impaired in the caudal hematopoietic tissue(CHT)of prpf31 knockout zebrafish.The emergence,migration,colonization,expansion,and maintenance of HSPCs were further observed by in situ hybridization and real-time fluorescent zebrafish imaging technologies.The results showed that the emergence,migration,and colonization of HSPCs were normal in prpf31 knockout zebrafish,but the prpf31 knockout zebrafish manifested severe defects in the expansion and maintenance of HSPCs in the CHT.The vascular development of prpf31 gene knockout zebrafish was observed by in situ hybridization and real-time imaging of fluorescent zebrafish.The results indicated that prpf31 gene knockout zebrafish showed normal vasculogenesis,and the vascular structure was intact,suggesting that the expansion and maintenance defects of HSPCs in the CHT during definitive hematopoiesis were possibly independent of the vasculogenesis and vascular structure of prpf31 gene knockout zebrafish.To conclude,the expansion,maintenance,and differentiation of HSPCs were specifically impaired in the CHT of prpf31 gene knockout zebrafish.To explore the molecular mechanism underlying defects in HSPCs expansion,maintenance,and differentiation upon prpf31 gene knockout,the apoptotic and proliferating status of HSPCs in the CHT were detected by fluorescence labeling.TUNEL staining showed that no excessive apoptotic HSPCs were observed in the CHT of prpf31 knockout zebrafish.Ed U staining and p H3 staining showed that prpf31 knockout did not influence DNA synthesis during S phase of mitotic HSPCs in the CHT,but resulted in M phase arrest of mitotic HSPCs in the CHT.Therefore,it was suggested that the M phase arrest of HSPCs was responsible for the defects in the development and differentiation of HSPCs.To clarify how prpf31 knockout caused M phase arrest of HSPCs,transcriptomic sequencing was conducted,and functional Gene Ontology enrichment analysis of the differential alternative splicing genes were performed in this study.The data revealed that in prpf31 gene knockout zebrafish,significantly aberrant alternative splicing genes were enriched in several mitosis-related terms,including chromosome organization,regulation of cell cycle,and microtubule-based process,etc.The differential alternative splicing events of the above three mitosis-related terms were confirmed by semi-quantitative reverse transcription PCR,which were almost consistent with the transcriptomic sequencing data.The results above suggested that prpf31 knockout lead to extensive abnormalities in the alternative splicing of mitosis-related genes,wich may be the cause of M phase arrest in HSPCs.The mitotic status of HSPCs was further observed by Tubulin staining.HSPCs in the CHT of prpf31 knockout zebrafish showed abnormal mitotic prometaphase morphology,with disorganized spindle microtubule arrangement and lower chromatin condensation.Moreover,four mitosis-related genes(septin6,smarcb1 b,tinf2,and usp22),which have been associated with the pathologies of specific hematologic diseases,and were confirmed aberrantly spliced,were found to be significantly downregulated in the CHT region of prpf31 knockout zebrafish by in situ hybridization.Thus,it was demonstrated that prpf31 may define the mitotic status of HSPCs by regulating the alternative splicing of mitosisrelated genes,and by which it participated in the expansion,maintenance,and differentiation of HSPCs.In conclusion,it is the first demonstration that Prpf31 is involved in the development of hematopoietic system,and the molecular mechanisms are explained.Prpf31 regulates the mitotic status of HSPCs by defining the alternative splicing of mitosis-related genes,and by which it participates in the expansion,maintenance,and differentiation of HSPCs.This study explores the new physiological functions of Prpf31,and provides a new cognitive perspective for clarifying the functional mechanism of splicing factors in the development of hematopoietic system. |
