Mechanistic Study Of How RBM4-regulated Splicing Inhibits Tumour Proliferation Through Hippo-YAP Pathway

Objective: Hippo-YAP pathway,a key pathway to regulate cell proliferation and organ size,has been reported frequently.The Hippo pathway has been discovered in Drosophila,and over the past ten years,its role of regulating organ size and cell proliferation has become increasingly clear.In mammals,the key components of the Hippo pathway include Mst1/2,WW45(also known as Sav1),LATS1/2,Mob1,TAZ,YAP,TEAD,etc.The transcriptional output of this pathway is mediated by TEAD proteins that partner with YAP to activate genes that stimulate cell proliferation.As one of the key effector of the Hippo pathway,YAP lacks DNA-binding motif,and thus recognizes its targets through interacting with TEAD proteins.In current model,Hippo signalling is mainlyregulated via protein phosphorylation and degradation.Intriguingly,some key components of the Hippo–YAP pathway undergo extensive regulation in RNA level through alternative splicing(AS),a major mechanism to expand coding capacity of human genome.In eukaryotes,gene expression is finely controlled by various aspects.Especially,the pre-RNA splicing plays an important role in the regulation of gene expression.It has been previously found that 98% of the human genome region is not encoding protein,and pre-RNA contains a great number of no-coding introns.One of the crucial steps inside this process is the pre-mRNA constitutive splicing during which intronic sequences are removed from the pre-mRNAs and exonic sequences joined to form the mature mRNA.Another level of regulation during this process is represented by alternative splicing.It's the main cause of protein diversity.The same pre-RNA can generate different mRNAs by using different alternative splicing modes,and then translates to different proteins.Abnormal alternative splicing can lead to dysfunction of many proteins.Therefore,alternative splicing is also closely related to the4 occurrence and development of many diseases,including tumor.Aberrant splicing is one of the most principal hallmarks of tumor,however the biological effects of splicing dysregulation,especially in the tumor,are rarely reported.Intriguingly,YAP and TEAD undergo extensive regulation in RNA level through alternative splicing.Consistent with our previous research,ectopic expression of the splicing factor RBM4 in tumors can promote TEAD4 to produce a short splicing isoform,TEAD4-S.The purpose of this study was to determine the role of TEAD4-S in cancer and how it affects the tumors through Hippo-YAP pathway.Methods: 1.PCR and Western blot were used to detect the expression of TEAD4-S in H157 cells and different tissues and organs.2.The cellular localization of TEAD4-S was examined by immunofluorescence assay.3.The stable cell line expressing RBM4 upon tetracycline induction was generated,and the effect of RBM4 on TEAD4 splicing in the stable cell line was tested using PCR and Western blot.4.A splicing minigene reporter was constructed.The specific binding site of RBM4 in the TEAD4 splicing regulation was determined by RIP and other experimental approaches.5.The interaction between TEAD4-S and YAP was measured by Co-IP method.6.The binding of TEAD4-S to downstream target gene was examined by ChIP assay.7.The effect of TEAD4-S on the interaction between YAP and TEAD4-FL was detected through IP assay etc.8.A luciferase gene driven by a TEAD-dependent promoter was applied to test if TEAD4-S antagonizes TEAD4-FL to repress YAP signaling.9.The effects of TEAD4-S on the proliferation and EMT were tested by using soft agar,colony formation,and western blot approaches in H157 and A549 cell lines.10.An animal model was established to verify the effect of TEAD4-S on tumorigenesis in vivo.Results: 1.TEAD4-S expresses in H157 cells and different tissues.2.TEAD4-S localizes in both cytoplasm and nucleus,and TEAD4-FL only localizes in nucleus.3.Overexpression of RBM4 promotes the production of TEAD4-S.4.RBM4 regulates the splicing of TEAD4 through CGGCCGG motif.5.The C-teminal of TEAD4-S has the function of interacting with YAP.6.The N-terminal of TEAD4-S loses the binding ability to target genes.7.The existence of TEAD4-S destroys the interaction ofTEAD4-FL and YAP.8.TEAD4-S antagonizes TEAD4-FL to repress YAP signalling.9.Overexpression of TEAD4-S can inhibit the proliferation and EMT of tumor cells.10.The overexpression of TEAD4-S can inhibit the growth of tumor in xenograft mice model.The expression of TEAD4-S is decreased in human tumors,and patients with high expression of TEAD4-S have a higher survival rate.Conclusions: TEAD4,the transcription factor that mediates Hippo–YAP signalling,undergoes alternative splicing facilitated by the tumour suppressor RBM4,producing a truncated isoform,TEAD4-S,which lacks an N-terminal DNA-binding domain,but maintains YAP interaction domain.TEAD4-S is reduced in cancer cells,and its re-expression suppresses cancer cell proliferation and migration,inhibiting tumour growth in xenograft mouse models.Furthermore,TEAD4-S is decreased in human cancers,and patients with elevated TEAD4-S levels have improved survivals.Altogether,these data reveal a splicing switch that serves to fine tune the Hippo–YAP pathway.