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. 2023 Dec 27;16(1):139. doi: 10.3390/cancers16010139

Figure 4.

Figure 4

RBM5 inhibits the expression of CARM1 via alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). (A) An RNA-binding protein immunoprecipitation (RIP) assay was performed using an RBM5 antibody to examine the interaction of RBM5 with CARM1 RNA. Upper panels: representative gel images; lower panels: western blot results. (B) The enrichment of the CARM1-coding sequence (CDS) RNA during the RIP assay was detected via RT–qPCR. (C,D) After transfection of T24 cells with LV-oeRBM5 and treatment with doxycycline (Dox), the levels of CARM1 mRNA and splice variants that lacked exon 9 (CARM1-Ex9-skip) were assessed using RT−PCR. GAPDH was considered the internal reference gene. (E,F) T24 cells were treated as described in (C), and western blotting was performed to examine the CARM1 protein expression. (G) Schematic of a Minigene splicing reporter. (H,I) RT−PCR analysis of the expression levels of CARM1 splice variants that lack exon 9 from Minigene splicing reporters (CARM1-E9M) coinfected with LV-shRBM5 and LV-oeRBM5, as described in (B). n = 4 biological replicates of RBM5-E6M. (J,K) RT−PCR analysis of the changes in expression of CARM1 splice variants that lack exon 9 following nonsense-mediated mRNA decay (NMD) inhibition via UPF1 depletion and Dox treatment. (L,M) CARM1 splice variants that lack exon 9 following NMD inhibition via cycloheximide (CHX) and Dox treatment. All data were derived from three independent experiments and expressed as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. their corresponding controls.