Elucidating the RNA-Protein Interactomes of Target RNAs in Tissue

Abstract

RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA-protein complexes.

Figure 1.. Overview of HyPR-MS procedure for tissue and capture analysis.

(A) Schematic showing tissue HyPR-MS workflow. (B) Purification of MALAT1 was verified using an RT-qPCR assay. Capture efficiency was calculated based on the depletion of MALAT1 between pre- and post-RNA capture samples. Capture specificity was calculated by dividing the C_t values of MALAT1 in the MALAT1 capture samples by the MALAT1 C_t values in the scrambled control samples. Figure created with Biorender.

Figure 2.. Polyadenylated RNA Capture in Mouse Liver.

(A) Overlap of identified proteins in polyadenylated RNA capture of three biological replicates. (B) Condensed list of GO terms enriched in the polyadenylated RNA capture of mouse liver (FDR <0.05). Figure created with Biorender.com.

Figure 3.. Proteins interacting with Polyadenylated RNA in Patient-derived Prostate Tumor Xenografts.

(A) Venn diagrams showing the overlap of identified proteins from the polyA RNA capture in the three biological replicates for each xenograft. (B-C) Condensed list of GO biological process and molecular function terms enriched in both XG147 and XG77 (FDR < 0.05). Figure created with Biorender.

Figure 4.. Analysis of Proteins Identified in MALAT1 RNA Capture Experiments.

(A) Table displaying identifications of proteins at 1% FDR. (B) Volcano plots displaying the distribution of all proteins for each xenograft with the significance level (negative log₁₀ p-value) plotted against the relative protein abundance (log₂ MALAT1 capture/scrambled control capture). Significantly changing proteins (p < 0.05, fold-change >2 or <−2) are highlighted in purple. Figure created with Biorender.