Functlonal Genomlc Screenlng And Characterization Of RNA-Binding Proteins In TGF-β Signaling

Transforming Growth Factor-β (TGF-β)superfamily controls a broad range of cellular responses in metazoan organisms, including cell proliferation and differentiation. Thus, dysregulation of TGF-βresponses has been implicatedin pathogenesis of human diseases, such as cancer. In the canonicalpathway, TGF-P binds toits receptors on the cell surface, and receptors are then activated to form a tetramer. This complex phosphorylates R-Smads to trigger subsequent signaling events. Phosphorylated R-Smads form a heteroligomeric (often trimeric) complex with Co-Smad. The activated Smad complex is then imported intothe nucleus and directly binds to the promoter regions of TGF-βtarget genes to regulate transcription.Regulation of TGF-P signaling at transcriptional level is well studied, while, yet it is still unclear whether and how TGF-P signalingundergoes post-transcriptional regulation. Upon transcription, precausor mRNAs must be processed to mature mRNAs by several steps, including alternative splicing. Most of post-transcriptional steps are controlled by RNA-binding proteins (RBPs). In order to search for important RNA-binding proteins involved in TGF-P signaling, we constructed an expression library of RBPs, and initially screened forRBPs in TGF-P signal transduction by reading their effects on TGF-P-induced SBE-luc reporter expression.Base on available genomic sequences, we compileda list of human RBPs, which has235RBPs containing one or more RNA-binding motifs. According to the Human ORFeome5.1cDNA library, we constructed expression plasmids harboring one of RBPs into the lentivirus vector using Gateway system. These proteins were expressed in HEK293T cells and then screened in HaCaT cells by SBE-luc reporter assay. We have found several important candidates, among which one candidate, designated RBP75, strongly promotedTGF-βsignaling. Further study showed that RBP75directly interacted with Smad2/3through its RNA recognition motifs. Importantly, ectopic expression of RBP75enhancedTGF-p-induced epithelial-mesenchymal transition (EMT) in epithelial cells MCF10A. These findings provide a novel mechanism underlying of TGF-β signaling byRBPs and implicate potential functions of RBPs in tumor metastasis.