Fig. 5: circTGFBR2(3-6) scaffolds IGF2BP3 and TGFBR1 mRNA.

A Identification of circTGFBR2(3-6)-binding proteins by RNA pull-down followed by mass spectrometry. The top statistically significant hits are shown. RNA-binding proteins are marked in red. B RNA pull-down analysis of circTGFBR2(3-6)-IGF2BP3 interaction in MDA-MB-231 cells. Western blotting with an IGF2BP3 antibody was used to detect IGF2BP3 in whole-cell lysates (Input) and immunoprecipitates (IP). C RNA immunoprecipitation (RIP) analysis confirming circTGFBR2(3-6)-IGF2BP3 interaction in MDA-MB-231 cells. RT-qPCR was performed to quantify circTGFBR2(3-6) levels in immunoprecipitates. Data are presented as mean ± SEM from three biological replicates, with significance assessed using an unpaired Student’s t-test. D RIP assay assessing TGFBR1 mRNA-IGF2BP3 interaction upon shRNA-mediated circTGFBR2(3-6) knockdown in MDA-MB-231 cells. RT-qPCR was performed to detect TGFBR1 in immunoprecipitates. Data represent mean ± SEM from five independent experiments, with significance assessed using two-way ANOVA followed by uncorrected Fisher’s LSD test. E Schematic representation of full-length TGFBR1 (FL) and its truncation mutants (F1, F2, F3, and F4). Locations of CDS and 3′UTR are indicated. F RIP assay evaluating the interaction between FLAG-TGFBR1 mRNA (FL and truncation mutants) and MYC-IGF2BP3 in HEK293T cells. RT-qPCR detected FLAG in immunoprecipitates. Data represent mean ± SEM from three biological replicates, with significance analyzed using two-way ANOVA followed by Šídák’s multiple comparisons test. G RNA pull-down assay assessing the interaction between FLAG-TGFBR1 mRNA (FL and truncation mutants) and circTGFBR2(3-6) in HEK293T cells. RT-qPCR detected FLAG in immunoprecipitates. Data represented as mean ± SEM from three biological replicates, with significance assessed using two-way ANOVA followed by Šídák’s multiple comparisons test. H Interaction between circTGFBR2(3-6) and either WT or MUT FLAG-TGFBR1-F1 in HEK293T cells, analyzed by RNA pull-down followed by RT-qPCR. Data represent mean ± SEM from three biological replicates, with significance assessed using a two-tailed unpaired Student’s t-test. I Schematic representation of FL IGF2BP3 and its truncation mutants (RRM12, KH12, and KH34). J Western blotting analysis of MYC-IGF2BP3 and its truncation mutants in HEK293T cells. GAPDH, loading control. RIP assay assessing the interaction between MYC-IGF2BP3 FL or truncation mutants and FLAG-TGFBR1-F4 (K) or circTGFBR2(3-6) (L) and in HEK293T cells. RT-qPCR detected FLAG in immunoprecipitates. Data are presented as mean ± SEM from three biological replicates, with significance analyzed using two-way ANOVA followed by Šídák’s multiple comparisons test. M Schematic working model for how circTGFBR2(3-6) binds to the KH12 di-domain of IGF2BP3 and promotes its interaction with TGFBR1 mRNA through the KH34 di-domain.