Fig. 6: The patient variant p.Arg836Gln disrupts numerous signalling pathways and leads to reduced SART3 RNA and protein levels.

RNA-seq and MS analysis of heterozygous, homozygous p.Arg836Gln variant or non-edited control iPSCs. a A Venn diagram of differentially expressed (DE) genes between groups (FDR < 0.05). b Number of DE genes is shown in blue for each comparison KEGG and Gene Ontology (biological process) pathway analysis identified 13 pathways/gene ontologies with significant enrichment (P < 0.05 adjusted for multiple comparisons, Benjamini Hochberg FDR) represented in the topmost DE genes between control and homozygous iPSCs (1000 genes based on FDR). c A schematic of the major spliceosome. SART3 (red) has been implicated in recycling U4 and U6 snRNAs. Components that are DE in variant iPSCs are shown (RNA-seq or MS. Bold text = both with change in same direction). d Intersection of the DE genes (FDR < 0.05) and proteins (FDR < 0.05) revealed a significant overlap (P < 10⁻⁹, one-sided Fischer’s Exact Test) with 350 genes/proteins DE in the same direction in both datasets (plotted as log₂ ratio/fold change). Genes/proteins with the highest upregulation (red) or downregulation (blue) are shown. SART3 is downregulated in the homozygous variant cells (green). e SART3 expression from RNA-seq (mean log₂ counts per million, CPM) *** = FDR = 1.52E-05 and 1.45E-04 respectively. Downregulation was confirmed by RT-qPCR – see Supplementary Fig. 6i. f MS found a significant difference in SART3 protein levels in homozygous iPSC line compared to control (***P = 3.77E-05), or heterozygous line (***P = 1.11E-04). g This was confirmed in western blot analysis – see also Supplementary Fig. 5p. h SART3 staining is nuclear in all three lines. RNA-seq and proteomic data analysis is provided in Supplementary Data 2.