PRPF40B regulates alternative splicing. (A,B) Schematic representation of the human Bcl-x and Fas minigenes and their derived splicing variants. Exons (boxes), introns (horizontal lines), and patterns of alternative splicing events (inclined lines) are represented. HEK293T cells were cotransfected with the Bcl-x (A) or Fas (B) minigenes together with an empty vector (Mock) or a PRPF40B expression plasmid (PRPF40B-OE) for overexpression experiments. For the RNAi experiments, HEK293T cells were cotransfected with the corresponding minigene together with siPRPF40B or siEGFP as a control. The graphs show the densitometric analysis results as the exon skipping average from three independent experiments (means ± SEM). (*) P < 0.05; (***) P < 0.005. A fraction of the cell lysates was analyzed by immunoblotting with the indicated antibodies to detect the PRPF40B and ERK2 proteins. (C,D) PRPF40B regulates the alternative splicing of the endogenous Fas gene. HEK293T cells were transfected with an empty vector (Mock) or a PRPF40B-expressing plasmid (OE). For the RNAi experiments, the HEK293T cells were transfected with siPRPF40B or siEGFP as a control. After total RNA extraction, RT-PCR (C) and RT-qPCR (D) were performed using the primers indicated in the schematic representations of the Fas gene. RT-PCR primers spanned the region between exons 5 and 7, including both the anti- and the proapoptotic isoforms of the Fas gene. RT-qPCR primers amplified the proapoptotic isoform including exon 6. The bar graphs represent the ratio of exon 6 inclusion, which is shown relative to the control that was set at 1. The data are from five independent experiments (means ± SEM). (*) P < 0.05; (**) P < 0.01. A fraction of the cell lysates was analyzed by immunoblotting with the indicated antibodies to detect the PRPF40B and ERK2 proteins.
