Splicing Factor SRSF1 Regulates The Production Of Wbp1l Alternative Spliceosome LncRNA-CEPT8 To Promote The Formation Of Chicken PGCs

Primordial Germ Cells(PGCs)are the primitive progenitor cells of germ cells in organisms,which are responsible for transmitting genetic between generations.The self-renewal and multi-directional differentiation potential are PGCs’ major biological properties,which can apply to the field of cell therapy,tissue repair,and even organ transplantation.The unique way of poultry PGCs migrate with the blood circulation improves the operability of their isolation and culture in vitro.This feature makes it have a wide range of potential applications in the production of transgenic chickens,the protection of endangered poultry germplasm resources,and somatic cell reprogramming.However,the number of PGCs that can be separated from a single chicken embryo is only 2000-5000,which is far from meeting the requirements for practical applications.For this reason,researchers tried to obtain a large number of PGCs by inducing differentiation in vitro,but due to the immaturity of the induction system,the problem of the limited number of high-quality chicken PGCs in vitro could not be solved.Therefore,in order to overcome the limitations of the application of chicken PGCs,it is urgent to clarify the mechanism of action of chicken PGCs.Studies have shown that the induction of paracrine signals,the inhibition of somatic cell fate,the change of epigenetic marks,and the maintenance of pluripotency play an important regulatory role in the formation of PGCs.With the rapid development of high-throughput sequencing technology in recent years,a growing number of factors have been confirmed to be involved in the regulation of germ cell formation:such as lncRNA and alternative splicing events.The previous research of our group found that lncRNA-CEPT8 plays an important role in the formation of chicken PGCs.Through bioinformatics analysis,it was found that lncRNA-CEPT8 was one of the members of Wbp1l alternative spliceosome.However,the mechanism of how Wbp1l forms lncRNA-CEPT8 through alternative splicing is not yet clear.Based on this,this study attempts to analyze the molecular mechanism of lncRN A and its regulation of PGCs formation from the perspective of alternative splicing.To this end,this study conducted an in-depth analysis of the sequence of lncRNA-CEPT8 and determined the key splicing factor SRSF1 that regulates its formation.Using molecular biology methods such as RIP and CoIP to explore how m6A affects SRSF1 to regulate the production of Wbpll alternative spliceosome lncRNA-CEPT8 to promote the formation of PGCs,systematically analyze the molecular mechanism of PGCs formation,provide technical support for improving the formation efficiency of PGCs in vitro,and lay a theoretical foundation for broadening the application scope of chicken PGCsThe research results are as follows:(1)lncRNA-CEPT8 is a key alternative spliceosome of Wbp1l in the formation of PGCs.Bioinformatics analysis found that lncRNA-CEPT8 and Wbp1l are located in the same region of the genome,and lncRNA-CEPT8 only contained Exonl and Exon2 of Wbp1l.Northern Blot and PCR amplification experiments confirmed that lncRNA-CEPT8 exists in PGCs.An overexpression vector with a full length of Wbp1l exon 3239bp was successfully constructed.qRT-PCR detection revealed that lncRNA-CEPT8 is the key alternative spliceosome of Wbp1l in the formation of PGCs.(2)SRSF1 is a key splicing factor in the formation of lncRNA-CEPT8.A total of three eligible splicing factors(RBP7,SRSF1,and MSI2)were obtained through bioinformatics prediction of lncRNA-CEPT8 Exon2.RNA-seq detected the expression of splicing factors in chicken ESCs,PGCs,and SSCs.The results showed that the expression levels of RBP7,SRSF1,and MSI2 were firstly increased and then decreased during the differentiation process from ESC to SSC,and SRSF1 was specifically highly expressed in PGCs.The qRT-PCR results showed that the expression trend of SRSF1 and RBP7 was consistent with lncRNA-CEPT8,while the expression level of MSI2 kept increasing during this differentiation process Western Blot showed that SRSF1 appeared at 32kD and was highly expressed in PGCs.RIP detection showed that SRSF1 could combine with Exon2 of Wbp1l in chicken PGCs.The CDS region of SRSF1 was cloned with a full length of 747 bp;The overexpression vector oeSRSF 1 of the splicing factor SRSF1 and the interference vectors shSRSF1-1,shSRSF1-2,and shSRSF1-3 were constructed respectively.Activity verification results showed that the oeSRSF1 vector can significantly increase the expression of SRSF 1(P<0.01),and shSRSF1-2 had the best interference effect(P<0.01).Therefore,shSRSF1-2 was selected for subsequent functional verification experiments.The function of SRSF1 on the formation of lncRNA-CEPT8 was verified in PGCs.The qRT-PCR results showed that overexpression of SRSF1 could significantly increase the expression of lncRNA-CEPT8(P<0.01),while interference with SRSF1 could significantly reduce the expression of lncRNA-CEPT8(P<0.01)without affecting the expression of Wbp1l.The above results confirmed that splicing factor SRSF1 was the key splicing factor in the formation of the Wbp11 alternative spliceosome lncRNA-CEPT8.(3)SRSF1 promotes the formation of chicken PGCs by regulating the production of lncRNA-CEPT8.Based on the in vitro BMP4 induction system,the interference vector and the overexpression vector of SRSF1 were transfected.Through cell morphology observation,it was found that after overexpression of SRSF1,EBs began to aggregate on the 4th day,and the number of EBs increased significantly on the 6th day.And after interference with SRSF1,EBs appearing on the 2nd day are smaller in shape and accompanied by a decrease in the number of cells,and a few EBs appeared until the 6th day.The results of qRT-PCR showed that compared with the BMP4-induced group,the expression of SRSF1 in oeSRSF1 group was significantly higher than that in the control group on the 6th day(P<0.01).The expression level of the C-kit was significantly increased on the 6th day(P<0.01).After interfering with the expression of SRSF1,Cvh and C-kit on the 6th day were significantly lower than the control group(P<0.05).Flow cytometric analysis was performed on cells induced to the 6th day in each group.The results showed that compared with the BMP4 group after SRSF1 interfered,the efficiency of PGCs formation in vitro was significantly reduced(P<0.05),while after overexpression of SRSF1,CVH-positive cells increased to 22.45%(P<0.05).After 0d chicken embryos were injected with PEI-encapsulated SRSF1 interference and overexpression plasmids in vivo,the qRT-PCR results showed that interference with SRSF1 can significantly reduce the expression of Cvh and C-kit(P<0.01),and overexpression of SRSF1 can significantly increase the expression of Cvh and C-kit(P<0.01).Flow cytometric analysis showed that compared with the control group(49.85%±2.05),the SRSF1 overexpression group showed a higher positive rate of CVH(60.55%±2.41),and after interference with SRSF1,the efficiency of PGCs formation in vivo(36.45%±2.61)significantly reduced(P<0.05).PAS staining was used to detect the changes in the number of PGCs formed in the genital ridges of each group.The results showed that compared with the control group,the number of PGCs in the genital ridge was down-regulated in the SRSF1 interference group The number of PGCs in the genital ridge of the SRSF1 overexpression group was increased.Further in vivo and in vitro experiments confirmed that SRSF1 can promote the formation of PGCs by regulating the production of lncRNA-CEPT8.The above results indicate that during the development of chicken PGCs,SRSF1 can play a key role by inducing the retention of Wbp1l Exon2 to produce lncRNA-CEPT8.(4)Study on the mechanism of m6A modification affecting SRSF1 binding to Wbp1l to regulate the formation of lncRNA-CEPT8.Bioinformatics analysis of Wbp1l Exon2 sequence obtained 5 potential m6A modification sites,and meRIP detection revealed that in PGCs,the region bound by SRSF1 on Wbp1l Exon2 also had a high enrichment of m6A,and there was no enrichment of m6A in ESCs.Exon2/4/5/7/8 of Wbp1l has extremely significant m6A modification in PGCs.To verify the function of m6A modification in the alternative splicing process of Wbp1l,the overexpression vector of m6A methyltransferase METTL3 was constructed.Based on overexpression of Wbp1l,the interference and overexpression vectors of Mettl3 were transfected respectively.The meRIP detection found that interference with Mettl3 significantly reduced the m6A methylation level on Wbp1l Exon2(P<0.01),while overexpressing Mettl3 significantly increase the level of m6A methylation on Wbp1l Exon2(P<0.01).The qRT-PCR results showed the interference of Mettl3 based on overexpression of Wbp1l significantly reduced the expression of lncRNA-CEPT8(P<0.01).Compared with overexpression of Wbp1l alone,the cooperative overexpression of Mettl3 promoted the expression of lncRNA-CEPT8(P<0.01).Neither overexpression nor interference with Mettl3 affected the expression of Wbp1l.The above results indicate that m6A modification participates in the formation of lncRNA-CEPT8 through METTL3.To further explore the mechanism of m6A influencing the alternative splicing process of Wbp1l,5 RRACH sites of Wbp1l Exon2 were progressively and cumulatively mutated,and 5 mutation vectors were constructed.meRIP results showed that the enrichment level of m6A on Exon2 was significantly decreased(P<0.01)when the mutation site 3 and 4 were gradually accumulated,and the expression level of lncRNA-CEPT8 was significantly decreased by qRT-PCR detection(P<0.01),indicating that m6A promotes the formation of the lncRNA-CEPT8.is a dose-dependent modification.The SRSF1 antibody was further used to perform RIP detection in each group.The results showed that when the 3rd and 4th positions were progressively mutated,the binding level of SRSF1 on Exon2 decreased significantly(P<0.01).The above results indicate that the m6A dose-dependently promotes the binding of SRSF1 and Wbp1l Exon2 to regulate the production of lncRNA-CEPT8.To verify whether METTL3 functions through direct binding with Wbp1l Exon2,RIP results showed that METTL3 could bind to Wbp1l Exon2.It has been found that methyltransferases(METTL3/14,WTAP,and KIAA1429)catalyzed the formation of m6A modification through mRNA in the form of a complex.In this study,String interaction analysis and CoIP experiments confirmed that METTL3/14 can interact with SRSF1 to affect the alternative splicing of Wbp1l.