SRSF3 Regulates The Alternative Splicing Of CTLA-4 Exon 3

Background:Cytotoxic T-lymphocyte-associated antigen-4(CTLA-4)is a negative immunoregulatory factor,which plays an important role in the regulation of immune response and the immunotherapy of tumors.Alternative Splicing of CTLA-4 exon 3 can produce two splicing isoforms: full length CTLA-4(flCTLA-4)and soluble CTLA-4(sCTLA-4),both of them can bind to the CD80 ligand and CD86 ligand on the surface of Antigen presenting cell(APC)to regulate immune response.Because of the late discovery of sCTLA-4,researches about CTLA-4 mainly focus on flCTLA-4,the mechanism and the function of sCTLA-4 have not been fully studied.However,many evidences show that sCTLA-4 participates in the immunoregulation of CTLA-4 and plays an important role in the process of immune response.At present,more and more studies are exploring the functional differences and mechanism of the two splicing isoforms produced by CTLA-4 exon 3 alternative splicing.However,the regulatory factors and mechanism of CTLA-4 exon 3 alternative splicing are still unclear.Serine/arginine-rich splicing factor 3(SRSF3)is an alternative splicing regulator with multiple functions.SRSF3 has also been proved to be a proto-oncogene and has been overexpressed in many tumors.Objective:The aim of this study was to explore the alternative splicing regulation mechanism of CTLA-4 eonx 3.Design and Methods:1.The genomic sequence of exon 1 to exon 4 of human CTLA-4 was amplified by PCR and linked to eukaryotic expression vector pEGFP-N1 to construct the wild-type minigene expressing exon 1 to exon 4 of CTLA-4.Analyzing the sequence of exon 3 of CTLA-4 by software and using CTLA-4 wild minigene as template,ten mutation minigenes of exon 3 of CTLA-4 were constructed by mutating three or four bases of exon3 into one unit.CTLA-4 wild-type minigene and mutant minigenes were transfected into293 cells respectively.The expression changes of two isoforms produced by alternative splicing of CTLA-4 exon 3 were observed by reverse transcription polymerase chainreaction(RT-PCR),and the key binding sites of CTLA-4 exon 3 alternative splicing were screened.2.CTLA-4 minigene expression plasmid was co-transfected with five alternative splicing factors predicted by the software by transfection technology for screening.The expression changes of two alternative splicing isoforms of CTLA-4 were observed by RT-PCR and the possible regulators of CTLA-4 exon 3 alternative splicing were screened.3.RNA interference technology was used to knock down the expression of SRSF3 protein and transfection technology was used to overexpress SRSF3 protein.The expression of two splicing isoforms produced by alternative splicing of CTLA-4 exon 3was observed by RT-PCR.The knockdown and overexpression of SRSF3 were verified by Western blot.Result:Alternative splicing of CTLA-4 exon 3 is closely related to SRSF3.When SRSF3 was overexpressed,the expression of flCTLA-4 decreased,while the expression of sCTLA-4 increased.When SRSF3 was inhibited,the expression of flCTLA-4 increased,while the expression of sCTLA-4 decreased.It is concluded that SRSF3 can promote the expression of sCTLA-4 by regulating the alternative splicing of CTLA-4 exon 3.At the same time,three key regulatory sites on exon 3 of CTLA-4 were screened: mt3 mutation site,mt5 mutation site,and mt9 mutation site(Fig.2.2).The binding site of SRSF3 regulating alternative splicing of CTLA-4 exon 3 may be located in mt5 mutation site,which needs further experimental verification.Conclusion:It is preliminarily proved that SRSF3 participates in the regulation of alternative splicing of CTLA-4 exon 3.It can inhibit the expression of flCTLA-4 and promote the expression of sCTLA-4.The binding site of alternative splicing of CTLA-4 exon3 regulated by SRSF3 may be located on mt5 mutation site,which needs further experimental verification.CTLA-4 antibody can effectively control the occurrence and development of tumors as an immune checkpoint inhibitor.while SRSF3,as an important proto-oncogene,provides a new direction for the immunotherapy of tumors by regulating the splicing of CTLA-4 exon 3.