Fig. 6

Knockdowns of CPSF3 or hnRNP UL1 and overexpression of TIA1 regulate AS during reprogramming. A Schematic representation of the experiments performed to study the effect of TIA1 overexpression / CPSF3 or hnRNP UL1 knockdown by RNA-seq. B TIA1-dependent events detected during reprogramming. The x-axis represents the ∆PSI value between Empty day 12 and day 0. The y-axis represents the ∆PSI value between T7-TIA1 day 12 and day 0 control. TIA1-dependent events (|∆∆PSI(T7-TIA1 − Empty)| ≥ 10) are represented by colored dots (palette representing the |∆∆PSI(T7-TIA1 − Empty)| value, n = 387). TIA1-independent events (|∆∆PSI(T7-TIA1 − Empty)| < 2) are represented by grey dots (n = 558). C CPSF3- and UL1-dependent events detected during reprogramming (left and right, respectively). The x-axis represents the ∆PSI value between shSCR day 12 and day 0 control. The y-axis represents the ∆PSI value between shCPSF3#1 or shUL1#1 day 12 and day 0 control. CPSF3/UL1-dependent events (∆∆PSI(average_shRNAs − shSCR) ≥ 10) are represented by non-grey-colored dots (palette representing the ∆∆PSI(average_shRNAs − shSCR) value). CPSF3/UL1-independent events (∆∆PSI(average_shRNAs − shSCR) < 2) are represented by grey dots. See Additional file 1: Figure S6C for ∆PSI values of the same events in shRNA#2 conditions. D Venn diagram representing the overlap between CPSF3-, UL1-, and TIA1-dependent events (left, the number of events in each category is shown). Barplot representing the percentage of CPSF3-, UL1-, and TIA1-dependent events which are also differentially spliced in B cell reprogramming (right, the percentage of overlap in each category is indicated). E Violin plots representing the distribution of PSI values of TIA1-dependent events in non-infected cells (NI, day 0) and day 12 cells infected with Empty or T7-Tia1 vectors. F Violin plots representing the distribution of PSI values of TIA1-independent events as in panel E. E,F Statistical significance was calculated by Fisher’s exact test comparing number of events with intermediate (25 < PSI < 75) or extreme PSI values (PSI ≥ 75 or ≤ 25) in each condition against Empty control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). See also Additional file 1: Figure S6D. G Boxplots representing the distribution of the indicated sequence features of TIA1-dependent exons, compared to TIA1-independent events and a random set of exons with intermediate PSI values not changing throughout reprogramming (Control CEx). Statistical significance was calculated using Matt, by paired Mann-Whitney U test comparing each condition to the Control CEx set (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). H RNA map representing the distribution of TIA1 binding motif in TIA1-dependent exons and flanking introns, compared to TIA1-independent and Control CEx. Thicker segments indicate regions in which enrichment of TIA1 motif is significantly different compared to Control CEx. I Gene Ontology (GO) terms enriched in genes containing TIA1-dependent events, compared to a background of all genes containing mapped AS events in the dataset. GO enrichment was calculated using GOrilla and GO terms were summarized for visualization using REViGO. The x-axis and the size of each bubble represent the −log10(p value) of each GO term. J Violin plots representing the distribution of PSI values of CPSF3-dependent events in non-infected cells (NI, day 0) and day 12 cells infected with shSCR or two shRNAs specific for CPSF3 (shC#1 and shC#2) or UL1 (shU#1 and shU#2). K Violin plots representing the distribution of PSI values of CPSF3-independent events as in panel J. J,K Statistical significance was calculated by Fisher’s exact test comparing number of events with intermediate (25 < PSI < 75) or extreme PSI values (PSI ≥ 75 or ≤ 25) in each condition against shSCR control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). See also Additional file 1: Figure S6E. L Boxplots representing the distribution of sequence features of CPSF3- and UL1-dependent exons, compared to the corresponding CPSF3- and UL1-independent events and Control CEx. Statistical significance was calculated using Matt, by paired Mann-Whitney U test comparing each condition to the Control CEx set (*, **, *** = p value < 0.05, 0.01, 0.001 respectively). M GO terms enriched in genes containing CPSF3- and UL1-dependent events, compared to a background of all genes containing mapped AS events in the dataset, performed as in panel I