Fig 2: Knockdown of RBM17, U2SURP, or CHERP Alters Expression of RNA-Processing Factors(A) Plot showing the overlap pattern of differentially expressed genes across the knockdown models of RBM17, U2SURP, or CHERP (n = 3 replicates/siRNA, including siScramble). The red and blue bars represent all genes common to the three datasets (from the three interacting proteins)and consistently altered in the same direction: downregulated (red) and upregulated (blue). The black dots beneath the x axis indicate which proteins have been knocked down for each dataset and the direction of the resulting gene expression changes. The percentages at the top of the graph describe the size of the group of overlapping changes relatively to the biggest and the smallest of the three datasets.(B) Validation of selected gene expression changes by qRT-PCR. The levels of selected transcripts were measured using qRT-PCR on samples from HEK293T cells treated with siRBM17, siU2SURP, or siCHERP and compared to cells treated with siScramble (n = 4 replicates/siRNA run in technical triplicates). Bars represent mean ± SEM; p value was calculated by two-tailed Student’s t test and significance was set at p < 0.05.(C) Network plot of functionally enriched categories in overlapping differentially expressed genes. Each node is a functional category. The width of the connector between nodes is directly proportional to the number of genes shared between respective categories.See also Tables S2 and S3.

Fig 3: Knockdown of RBM17, U2SURP, or CHERP Reveals Shared Splicing Changes Affecting Numerous RNA-Processing Factors(A) Number of exclusion and inclusion splicing events per RNA sequencing dataset relative to the five categories of AS evaluated by rMATS (Shen et al., 2014). The fourth column describes the size of the list of shared events (exclusions and inclusions) across the three datasets calculated as absolute number and as relative percentage of the dataset with the smallest number of events.(B) Number of cryptic junction gains and losses per RNA sequencing dataset, calculated using CrypSplice. The last column indicates the size of the list of shared events across the three datasets calculated as absolute number and as relative percentage of the dataset with the smallest number of events.(C) Network plot of functionally enriched categories in overlapping differentially spliced genes. Each node is a functional category. The width of the connector between nodes is directly proportional to the number of genes shared between respective categories.See also Figures S5–S7 and Tables S4, S5, S6, and S7.

Fig 4: RBM17, SF3B1, U2SURP, and CHERP Interact with One Another and Regulate Reciprocal Protein Stability(A–C) Representative western blots (left) and protein-level quantifications (right) of SF3B1, U2SURP, CHERP, and RBM17 in HEK293T cells treated with siSF3B1 (A), siU2SURP (B), or siCHERP (C) and compared to cells treated with control siScramble (n = 6–18 replicates/siRNA). All data were normalized to VCL, used here as loading control. Bars represent mean ± SEM; p value was calculated by two-tailed Student’s t test, and significance was set at p < 0.05. The presence of a dashed line between Scramble and siRNA samples indicates that the image of the membrane has been modified to eliminate a marker lane between the two sets of samples. See also Figures S3 and S4.