Studies Of The Functions Of DMTF1 Isoforms And The Mechanism Of Their Alternative Splicing In Breast Cancer

The DMTF1 gene located on chromosome 7 is a haplo-insufficient tumor suppressor.The DMTF1 pre-mRNA with 18 exons can produce three mRNA isoforms,indivadually encoding DMTF1α,β and y proteins,through alternatively splicing.Among these three protein isoforms,DMTF1α plays a tumor suppressive role,β promotes oncogenesis,but the function of y remains largely undetermined.In cancer cells,aberrant alternative splicing can facilitate tumorigenesis through both upregulating oncogenic transcript expression and downregulating normal transcripts.Previous studies indicated that splicing decisions of DMTF1 pre-mRNA are aberrantly altered in approximate 30%of breast cancer,and therefore increased DMTF1βprotein levels and reduced α levels.Immunohistochemical results revealed that DMTF1β levels was increased in 60%samples of breast cancer patients.Meanwhile,DMTF1β expression levels correlated with poor prognosis of breast cancer patients.However,to date,the molecular mechanism regulating alternative splicing of DMTF1 pre-mRNA has not been determined.The results of the current study are as follows:1.Flow cytometric analyses,RNA interference,RT-PCR,Western blot and RT-qPCR studies demonstrated that both the RNA and protein stability of DMTF1β and γ isoforms are lower than that of DMTF1α.2.Immunofluorescence assays revealed that all three DMTF1 isoforms were localized in nuclei,and two basic residues,K52 and R53,in the DMTF1 isoforms were crucial to their nuclear localization.3.Luciferase reporter assays,RT-qPCR,electrophoretic mobility shift assays(EMSA)and coimmunoprecipitation showed that both DMTF1β and γ could interact with DMTF1α and antagonize its function of transcriptional activation.Furthermore,Kaplan-Meier analyses revealed that the ratios of both DMTF1β/α and γ/α significantly correlated with poor prognoses of breast cancer patients,and scratch assay indicated that ectopic introduction of DMTF1β and y promoted migration of breast cancer cells.4.Minigene reporter assays,cross-linked immunoprecipitation followed by qPCR(CLIP-qPCR),RT-qPCR,RNA interference-mediated SRSF5 knockdown and bioinformatics analyses demonstrated that highly expressed SRSF5 protein could bind to the splicing enhancer elementβGAA,interfere with the recognition of SF1 in the DMTF1 intron 9,and potentially change RNA structure of that region,leading to increased splicing of DMTF1β and γ.5.Dual-enzyme splicing reporter assays,RT-qPCR and Western blot studies revealed that DMTF1α,β and γ proteins mediated their own pre-mRNA splicing by negative feedback regulation,and consequently favor DMTF1β and γ splicing.Moveover,CLIP-qPCR,EMSA and mutated dual-enzyme splicing reporter assays identified a DMTF1 binding site in the DMTF1 intron 9,which served as a regulatory element of DMTF1α,β or γ in their negative feedback loops.6.Molecular docking prediction,DMTF1α mutation studies,dual-enzyme splicing reporter assays and EMSA assays were employed to demonstate that DMTF1α,β and y proteins contained a shared nucleic acid binding region(NBR),and the residue R212 was a crucial amino acid for the function of the NBR.Meanwhile,cell proliferation assays showed that its nucleic acid binding affinity is essential for the tumor suppressive activities of DMTF1α.In summary,this study determined the potential oncogenic activities of DMTF1β and γ in cancer development,discovered a new characteristic of the three DMTF1 isoforms in binding RNA and regulating the splicing process,innovatively delineated the molecular mechanism regulating the alternative splicing of DMTF1 pre-mRNA,and identified SRSF5,as a splicing factor,promoted the splicing of DMTF1β and γ.These experimental data filled the gap in the understanding of mechanisms underlying the alternative splicing of DMTF1 pre-mRNA,improved our knowledge of the functional roles of the three DMTF1 splicing isoforms during oncogenesis,and provided insights to the developing novel anticancer therapeutic strategies through modulating alternative splicing processes.