Role And Mechanism Of Splicing Factor Bud31 In Mouse Spermatogenesis

The process of spermatogenesis is complex and involves multiple biological events,including spermatogonia mitosis,spermatocyte meiosis,and mature spermatogenesis.The process of spermatogenesis is finely regulated by a variety of factors,but the mechanism of action is still unclear.Therefore,in-depth investigation of the molecular mechanism of spermatogenesis will provide a theoretical basis for effective treatment of infertility.RNA splicing is an important part of gene expression regulation in eukaryotes.The spliceosome processes precursor RNA,removes introns and connects exons.Alternative splicing(AS)is the process of selecting different splicing sites to produce multiple products in the process of pre-mRNA splicing,which is an important mechanism for generating gene product diversity.AS occurs in about 60%of mouse genes and 95%of human genes.Studies have shown that the abnormality of alternative splicing is closely related to the occurrence of many diseases.AS plays an important role in cell differentiation and ontogeny and is precisely regulated,showing spatiotemporal specificity and tissue specificity.The alternative splicing events that occur in brain and testicular tissues are more complex than in other tissues.The mechanism of alternative splicing regulation during spermatogenesis is still not fully elucidated.Bud31,as a component of spliceosome in yeast,is involved in the regulation of spliceosome assembly and catalytic activity.Bud31 deletion leads to defects in budding formation and disorder of actin distribution in yeast cells.BUD3 1 is a synthetic lethal gene of MYC in human mammary epithelial cells,and knockdown of BUD31 in tumor cells with overactivation of MYC leads to apoptosis.There are few studies on the function of Bud31 in physiological state.In this paper,we focused on the function and mechanism of Bud31 and its regulation of alternative splicing in spermatogenesis.In this paper,we constructed a conditional Bud31 knockout mouse model using CreLoxp system,and then constructed Bud31 specific knockout mice at different developmental stages using Vasa-Cre(E15.5)expressed by early spermatogenic cells and Stra8-GFPCre(P7)expressed by late spermatogenic cells.In addition,Stra8-GFPCre mice and Bud31flox/flox mice were used to construct generalized Bud31 knockout(Null)mice.Based on the above Bud31 gene knockout mouse model,we investigated the role and mechanism of splicing factor Bud31 and its regulated splicing molecular network in mouse spermatogenesis,and obtained the following results.1.Splicing factor Bud31 is a key splicing factor regulating the fertility of male mice(1)Analysis of Bud31 expression in mouse testis at different developmental stages:Firstly,we used Evo-Devo Mammalian organs database to analyze the RNA-seq data of testicular tissues of mice at different developmental stages(E18.5,P0,P3,P14,P28 and P63).We found that the expression levels of genes involved in regulating alternative splicing events were significantly increased on the third day after birth.Through the analysis of the expression level of 134 core splicing factor,found that 10 splicing factor in mice after the birth of the expression level increased significantly,the expression level of Bud31 significantly raised three days after he was born in mice,and associated with the expression pattern of splicing events,further analysis found that Bud31 proteins are mainly located in the nucleus of male mice reproductive cells.(2)The generalized Bud31 knockout(Null)mice constructed from Stra8-GFPCre mice and Bud31flox/flox mice could survive normally,but the Null mice had significant weight loss,small testicular size and infertility.These results suggest that Bud31 may play an important regulatory role in ontogeny and spermatogenesis of mice.(3)Germline specific homozygous knockout mice were obtained by mating Bud31flox/flox mice with Vasa-Cre(E15.5)mice.Phenotypic analysis showed that adult Bud31-vKO mice were sterile,had significantly reduced testis and lost germ cells,presenting "sertoli cell only syndrome".The percentage of spermatogonia(THY1+/c-kit-)was analyzed by flow cytometry from day 1 to day 5 after birth.It was found that the number of spermatogonia in knockout mice decreased significantly from day 4 after birth.Immunofluorescence co-staining of Bud31 and Plzf(marker of spermatogonial stem cells)also showed that the number of spermatogonial stem cells in Bud31 knockout mice decreased significantly on postnatal day 4.Immunofluorescence co-staining of Lin28a and Plzf also further confirmed that Bud31 knockdown resulted in a significant reduction of spermatogonial stem cells.Microcellular RNA sequencing(Smart-seq)was used to analyze the transcriptome of Bud31 knockout and control mice(P4)germ cells.It was found that AKT-mTOR signaling pathway was significantly enriched and decreased.These results suggest that Bud31 plays an important role in the maintenance of spermatogonia homeostasis in mice.Loss of Bud31 leads to the arrest of self-renewal and differentiation of spermatogonia stem cells,resulting in spermatogenesis disorder and male sterility.2.Bud31(Stra8-GFPCre)knockout caused failure of normal initiation of meiosis in male mice(1)To explore the effect of Bud31 on meiosis,Stra8-GFPCre(P7)mice were mated with Bud31flox/flox mice to obtain germline homozygous knockout mice.Phenotypic analysis showed that the testis of Bud31-sKO mice were significantly smaller than those of control mice,and the seminiferous tubules had no spermatogenic cells.Only a few spermatocytes in the preleptotene stage were observed.(2)Bud31-sKO mice and control mice(P12)were selected and stained with Sycp3 and yH2AX by chromosome laying and immunofluorescence.The results showed that only the spermatocytes in the preleptotene stage were found in Bud31-Sko mice,while a large number of spermatocytes in the conjutene stage were found in the control mice.Rtqpcr was used to detect the mRNA expression of spermatogonia differentiation related genes(Stra8 and C-Kit)and meiosis related genes(Dmc1,Sycp3,Sycpl and Rec8).The results showed that Bud31 knockdown significantly down-regulated spermatogonia differentiation related genes such as C-Kit and meiosis related genes such as Dmcl.These results indicated that Bud31 knockdown caused failure of normal initiation of meiosis in male mice.(3)To investigate whether Bud31 expression is regulated by retinoic acid(RA),The primary testicular cells of wild-type mice(P10)were treated with RA(0.5μm),RA inhibitor WIN 18,446(0.5 μm),and RAR inverse agonist BMS493(10 μm)for 16 h.The expression levels of Bud31 and RAR(Stra8,Hoxa1,Rarb and Rarg)were detected by RTqPCR and Western blotting,respectively.The results showed that RA increased the expression of RAR,but Bud31 mRNA and protein levels did not change.RA inhibitor WIN 18,446(0.5μm)and RAR antagonist BMS493(10 μm)showed that RA inhibition did not reduce the mRNA and protein levels of Bud31.These results indicated that Bud31 expression was not regulated by RA,and Bud31 regulation of meiosis was independent of RA.3.Bud31 regulates the molecular network and target genes of alternative splicing in mouse spermatogenic cells(1)Co-IP mass spectrometry showed that Bud31 binds to several splicing factors,including SF3B1 and HNRNP proteins,further suggesting that Bud31 may participate in spermatogenesis by regulating alternative splicing in mice.(2)Using Smart-seq data to analyze splicing expression profiles,we found that Bud31 knockdown resulted in many splicing events,among which exon hopping(57%)and intron retention(20%)were the main splicing types regulated by Bud31.Bud31 knockdown resulted in a decrease in the number of exons and an increase in the number of introns retained throughout the genome,and a decrease in the efficiency of 5’ and 3’splicing.RIP-seq analysis of mouse primary germ cells showed that Bud31 mainly bound to introns,especially at the junction of RNA exons and introns,and two highly conserved binding motifs were identified.(3)In order to further explore the alternative splicing molecular network and target genes regulated by Bud31,the Smart-Seq and RIP-seq data were integrated and analyzed,and 392 target genes that both undergo alternative splicing and bind to Bud31 were found.Twelve down-regulated genes,including Cdk2,were selected as candidate target genes regulated by Bud31.(4)Cdk2 knockout mice can survive normally but are sterile.Cdk2 deletion leads to the arrest of spermatogonia self-renewal and differentiation.Therefore,we took Cdk2 as an example to further explore the mechanism of Bud31 regulating target gene alternative splicing.Using RIP-PCR,splicing reporter gene,RNA pull-down and other experimental methods,it was found that germline specific knockdown of Bud31 resulted in the increase of the retained splice isoforms of Cdk2 gene intron 1,the decrease of classical splice isoforms,and the decrease of Cdk2 protein level,which eventually led to the self-renewal block of spermatogonia stem cells.Meiosis fails to initiate properly.In summary,the conclusions of this paper are as follows:1.Bud31 knockout leads to the arrest of self-renewal of spermatogonia stem cells,the disappearance of spermatogonia,and finally the male sterility of mice;2.Bud31 knockout caused the failure of normal initiation of meiosis and meiosis arrest in pre-leptotene phase in male mice;3.Bud31 regulates many alternative splicing events in mouse spermatogenic cells,among which exon hopping and intron retention are the main splicing types regulated by Bud31.4.Bud31 participates in spermatogenesis by regulating the alternative splicing network of many genes,including Cdk2.