Fig. 4: Determination of the non-poly(A) RNA-bound proteomes of mammalian organs.
From: The RNA-binding protein landscapes differ between mammalian organs and cultured cells
a Schematic representation of non-poly(A)RIC. eRIC supernatants are depleted of poly(A) RNA by oligo(dT) bead selection and hence predominantly contain non-poly(A) RNA and non-poly(A) RNP complexes that were purified by modified 2C45. Created with BioRender.com. b Volcano plots showing significant enrichment of RBPs in UV crosslinked over non-irradiated samples. Red dots, hits: FDR <0.05, FC >2. Blue dots, candidates: FDR <0.2, FC >1.5 (moderated two-sided t-test with FDR multiple testing correction). c Venn diagram showing the number of shared and organ-specific non-poly(A) RBPs identified. d Gene Ontology (GO)-term enrichment analysis (Fisher’s one-tailed test with g:SCS multiple testing correction). Selected GO terms corresponding to molecular function are displayed (see Supplementary Data 9 for the full list of GO terms). e Hierarchical clustering and heatmap of the poly(A) and non-poly(A) RBPs identified in the brain, kidney, and liver, shown as the Log.2 ratio of protein abundance in irradiated versus non-irradiated samples. f Venn diagram depicting the number of proteins interacting exclusively with poly(A) RNA or non-poly(A) RNA or with both biotypes of RNA. High-confident poly(A) RNA binders (in yellow) are defined as the eRIC hits that were not detected neither in the non-poly(A) RNA-bound proteomes described here nor in an in-depth analysis of non-poly(A) RNA binders performed in the liver (see below). g Normalized signal sum of identified poly(A), non-poly(A), and dual RBPs, as appropriate, in ex vivo eRIC eluates (first and fourth panels) and non-poly(A)RIC (middle two panels). Protein signal was adjusted by RNA content (Fig. 3e). Blue, brain; red, kidney; orange, liver. Center lines indicate the median, box borders represent the interquartile range (IQR), and whiskers extend to ±1.5 times the IQR; outliers are shown as black dots. For each organ, four +UV eRIC and four +UV non-poly(A)RIC eluates were generated, each derived from a single mouse; eRIC and non-poly(A)RIC eluates from two mice were combined, rendering n = 2. Organ sections from four mice were pooled to generate one -UV eRIC and one non-poly(A)RIC eluate per organ (n = 1). **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001, n.s.: not significant (one-way ANOVA with Tukey post hoc test).
