Fig 1: siRNA-mediated hnRNPL reduction causes AS changes that partly mirror those elicited by CHD8 suppression. (A) The bar graph reports the normalized expression levels (2-??CT) of hnRNPL transcript following Si-C administration for 72 h in hiNPCs. Four different biological replicates per experimental condition are reported. Means ± SE are shown. One-tail t-test was performed, with ***P =0.001. (B) Representative western blot images illustrate total hnRNPL levels, comparing hiNPC exposed to siRNA against hnRNPL (Si-C) and control (Si-Scr). Two representative biological replicates per condition are presented. Comparable amounts of total protein were loaded as detected by the HSP90 loading control. (C) The bars in the chart represent normalized hnRNPL protein levels comparing hiNPCs exposed to siRNA against hnRNPL (Si-C) and control (Si-Scr). HnRNPL bands were normalized on HSP90 loading control. Mean values ± SE from four independent biological replicates are plotted. T-test for two mean populations was performed, *P =0.05. (D) Venn diagrams represent the overlap between genes presenting aberrant splicing following CHD8 suppression and genes presenting altered AS events after siRNA-mediated reduction of hnRNPL (72 h post-electroporation). The AS events are detected by SUPPA. The number of genes for each condition is indicated. The enrichment significance for the intersection is computed by Fisher's exact test and represented by colors. Color-coded key: –log10(P-value). The P-value and odds ratio are reported. (E) The stacked bar plot represents the 473 differential AS events detected by SUPPA in hiNPCs subjected to siRNA against hnRNPL distributed by event type. Event types: SE, skipped event; RI, retained intron; MX, mixed event; A3, alternative 3'; A5, alternative 5'; AF, alternative first exon; AL, alternative last exon. (F) The bar plot represents GO biological process and KEGG pathway terms significantly enriched in genes presenting altered AS events as detected by SUPPA following CHD8 and hnRNPL suppression (intersection in D). The bars are ordered according to adjusted P-values in –log10 scale; the x-axis represents the number of genes enriched for each term. (G) CHD8 functions at transcription initiation, elongation and regulation of AS. Schematic representation of the molecular mechanisms proposed to explain CHD8’s roles at promoters/enhancers and in the modulation of AS (figure created in BioRender.com). CHD8 interacts with hnRNPL, possibly through stabilizing RNA bridges (red hairpins). Thus, CHD8/hnRNPL association, possibly also recruiting the Mediator complex (106), might stabilize enhancer/promoter looping and transcriptional initiation. However, hnRNPL also solidly interacts with SETD2 at elongating RNAPII, thus implicating CHD8 in the regulation of RNA processing and AS. By comparing CHD8 and SETD2 MS experiments [our data and (39)], a number of other hnRNP interactors emerge, providing a functional link between SETD2, hnRNPs and CHD8 with elongating RNAPII, with functional consequences for the regulation of the splicing machinery. Differential alternative splicing events can be modulated by the CHD8/hnRNPL/SETD2 complex.