Fig. 6

The exploration of the binding region of TPM3P9 and RBM4. a Diagram of plasmids encoding GFP-tagged full-length RBM4 (RBM4-GFP) and GFP-tagged RBM4 truncations (RBM4-N-GFP and RBM4-C-GFP). The diagram was edited using Adobe Illustrator. b The indicated GFP-tagged wild-type RBM4 and mutant RBM4 plasmids as well as the TPM3P9-Flag plasmid were transfected into HEK293T cells, and co-immunoprecipitation with an antibody specific to GFP revealed that RBM4-GFP and RBM4-N-GFP, but not RBM4-C-GFP, were capable of binding to TPM3P9. The black arrows represent the destination blots. c The indicated GFP-tagged wild-type RBM4 and mutant RBM4 plasmids as well as the TPM3P9-Flag plasmid were transfected into HEK293T cells, and coimmunoprecipitation with an antibody specific for Flag revealed that RBM4-GFP and RBM4-N-GFP but not RBM4-C-GFP were capable of binding to TPM3P9. The black arrows represent the destination blots. d Diagram of plasmids encoding the RBM4 N-terminal truncations lacking RRM1 (N1-GFP), RRM2 (N2-GFP), or both RRM1 and RRM2 (N3-GFP). The diagram was edited using Adobe Illustrator. e, f The indicated GFP-tagged N-terminal domain deletion mutants of RBM4 were co-transfected with TPM3P9-Flag into HEK293T cells, and co-immunoprecipitation was performed using an antibody specific for Flag (e) or GFP (f); the results showed that both N1-GFP and N3-GFP, without the RRM1 motif, could not bind to TPM3P9. g RNA immunoprecipitation (RIP) showing the binding ability of different RBM4 truncations to TCF7L2 pre-mRNA. h Crosslinking immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) analysis of RBM4 revealed that RBM4 specifically crosslinked to an intron upstream of exon 13 in TCF7L2 pre-mRNA. i The minigene assay and RIP assay using a binding sequence mutant with a mutation in the intron upstream of exon 13 confirmed that RBM4 bound only to the wild-type TCF7L2 pre-mRNA