Fig 1: RNA G4s in ribosomal protein mRNA regulate translation. (a) Example snapshots demonstrating BG4 peaks in 5′ UTRs and overlapping iCLAE peaks. BG4 track depict logFC of BG4 IP vs input and iCLAE track depict counts per million. (b) Western blot analysis of ribosomal protein levels following shRNA mediated depletion of DDX3X, DHX36 and GRSF1. Actin serves as loading control. Number below each lane represents fold change in ribosomal protein levels compared to non-targeting control (NTC) when normalised to actin. (c) Fold change in luminescence comparing in vitro translation of luciferase gene from wild-type ribosomal protein UTRs containing G4 (UTRQ) to mutant UTRs with G4 disruption (MutQ). (d) UV absorption at 254 nM following sucrose fractionation for polysome profiling of cells treated with DMSO (black) and 2 μM PDS (blue) for 45 min. Monosomal and polysomal fractions are highlighted. (e) Fold change in translation efficiency (logFC TE) of each transcript plotted against the significance (− log10 p-value) over three independent biological replicates. All ribosomal protein mRNAs are highlighted in red and those with significant changes (FDR < 0.1) are labelled with the corresponding protein names.
Fig 2: RNA binding proteins interact with G4s in mRNA. (a,d) Incidence of iCLAE peaks within protein-coding, non-coding or unannotated regions (other) of the genome. (b,e) Enrichment of iCLAE peaks at annotated RNA features. (c,f) Enrichment of iCLAE peaks at predicted G4 motifs. Enrichment calculated following random shuffling of peaks in the transcriptome (FDR = 1 × 10−4). Error bars represent 95% confidence interval. (g–i) Distribution of DHX36, DDX3X and GRSF1 iCLAE reads relative to G4 motifs. Graphs denote pileups of iCLAE reads from multiple sites in CPM. DDX3X iCLAE data has been previously published11.
Fig 3: Identification of cellular G4s using BG4 uvRIP. (a) Experimental strategy. (b) Example snapshot of BG4 uvRIP peak in the 5′ UTR of E2F4 gene. A negative control is provided by uvRIP using a non-G4 specific A9 antibody. (c) Overlap of BG4 uvRIP peaks with protein-coding, non-coding or unannotated regions of the genome. (d) Enrichment of BG4 uvRIP peaks within annotated RNA features. (e) Enrichment of G4 motifs within BG4 uvRIP peaks. (f) Enrichment of BG4 uvRIP peaks within iCLAE sites for DDX3X, DHX36 and GRSF1. For (d–f) enrichment calculated following random shuffling of peaks in the transcriptome. (g) Percentage overlap of BG4 uvRIP peaks with iCLAE datasets for DDX3X, DHX36 and GRSF1.
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