Fig. 1: A method for the specific determination of the 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 ex vivo eRIC (enhanced RNA interactome capture) applied to organs. Intact flash-frozen organs are sectioned into 30 µm slices amenable for UV irradiation. Following UV crosslinking (indicated by a red dot), tissue sections are lysed under denaturing conditions. RNA-binding proteins (RBPs) bound to polyadenylated RNA are subsequently isolated under highly stringent conditions using an LNA-modified oligo(dT) probe coupled to magnetic beads5,9. A fraction of the isolated material is used for RNA analysis. The rest is subjected to RNase digestion to retrieve RBPs. Following solid-phase-enhanced sample preparation (SP3)54,55, peptides subjected to tandem mass tag (TMT) labeling are multiplexed and analyzed using LC-MS/MS (liquid chromatography/tandem mass spectrometry). Created with BioRender.com. b–e ex vivo eRIC was used to characterize the RNA-bound proteomes of the brain, kidney, and liver from adult C57BL6/J mice. b RT-qPCR analysis of 18S rRNA as well as Actb and Gapdh mRNA abundance in eRIC eluates versus input, demonstrating enrichment of mRNA. Values are expressed relative to the respective input (input mean corresponds to 1.0). c qPCR analysis of mRNA versus genomic DNA (gDNA) for the housekeeping genes Actb and Gapdh, showing that gDNA contamination is minor. b, c n = 4 biologically independent experiments. d 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). The combined ex vivo eRIC data from the brain, kidney, and liver reveal more than 1300 hit RBPs (see Supplementary Data 1). e Scatter plots comparing the normalized signal sums in ex vivo eRIC eluates obtained from independent experiments performed with distinct animals. Pink dots, proteins harboring known RNA-binding domains (see section “eRIC uncovers organ RBPs not previously detected in cultured cells”). d, e for each organ, four +UV eRIC eluates were generated, each derived from a single mouse; eRIC eluates from two mice were combined, rendering n = 2. Organ sections from four mice were pooled to generate one -UV eRIC eluate per organ (n = 1). Source data are provided as a Source Data file.
