Fig 2: SNPs in the 3’UTR MRE modify miRNA-mediated regulation of target genes. (A) Flowchart describing steps utilized to prioritize candidate miRNA-MRE interactions for further study. Of the 24 candidates selected, 11 SNP-resident MREs are in the 3’UTR and 13 SNP-resident MREs are in the CDS of target genes. (B) Schematic describing how a SNP in a MRE could enhance or disrupt miRNA-mediated target gene regulation. (C–H) HEK293 cells were transfected with constructs containing the reference SNP (Ref), the alternative ‘candidate’ SNP (Alt), or a scrambled MRE (Scr) for the following genes (C) RAPGEF1, (D) SOD1, (E) CARF, (F) TUBB4A, (G) FBXO45 and (H) RPRD1A and analysed for RNA and protein expression. RNA and protein was harvested 48 h post-transfection. Left panel: RT-qPCR for Renilla (normalized to Firefly mRNA), presented as fold change relative to WT (n = 9). Right panel: Relative luminescence units (RLU) normalized (Renilla/Firefly) and presented as fold change relative to WT (n = 12). All panels: data presented as box & whiskers plot (min to max) and analysed by one-way ANOVA with Sidak’s multiple comparison test, **P < 0.01, ***P < 0.001.

Fig 3: SNPs in the MRE alter miRNA binding in iPSCs. Three altSNP+/- iPSC lines for each of the 6 genes were left untreated (Ut) or transfected with the non-targeting (Nt) control or respective miRNA reagents (RAPGEF1:anti-miR-124-3p, SOD1:anti-miR-139-5p, CARF: mimic of miR-181-5p, SREBF1:anti-miR-29b-3p, ABCA7:anti-miR-34a-5p, PICALM: anti-miR-34a-5p + anti-miR-103a-3p). Ago2 immunoprecipitation (anti-Ago2 Ab) and associated RNA was harvested 24 h later. RT-qPCR using allele-specific primers (Ref vs Alt) for respective genes (normalized to HPRT and total gene expression Ref + Alt) was performed. iPSC lines used (reference Figure S6): RAPGEF1 (2,13,14); CARF (1,6,12); SOD1 (4,10,12); SREBF1 (4,5,6); ABCA7 (1,7,10); PICALM (3,7,11). Data are presented as the ratio of SNP/WT (n = 12). Data are presented as box & whiskers plot (min to max) and analysed by one-way ANOVA with Sidak’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001.