Abstract

Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and non-coding RNAs are widely detected EV cargo, and RNA-binding proteins (RBPs), such as hnRNPA2B1, have been circumstantially implicated in sorting vesicular RNAs. However, the contribution of competitive RBP-RNA interactions responsible for RNA-sorting outcomes still needs to be deciphered, especially for EV-RNA interference and predictability. We conducted a reverse proteomic analysis that prioritized heterogeneous nuclear ribonucleoproteins recognizing purine-rich RNA sequences representing a subset of previously identified EXO motifs. A screening campaign using a full-length human hnRNPA2B1 protein and artificial purine-rich RNA brought to small molecule inhibitors orthogonally validated through biochemical and cell-based approaches. Selected drugs effectively interfered with a post-transcriptional layer impacting secreted EV- RNAs, reducing the vesicular pro-inflammatory miR-221 while counteracting the hnRNPA2B1- or TDP43Q331K-dependent paracrine activation of NF-{kappa}B in EV-recipient cells. This study demonstrates the possibility of predicting the EV-RNA quality for developing innovative strategies targeting discrete paracrine functions. SummaryExtracellular vesicles (EVs) are cell-released, heterogeneous lipid particles conceived as vehicles for intercellular communication. RNA is a widely detected cargo, and the comprehension of EV-sorting mechanisms represents a step forward in predicting EV quality and associated paracrine effects. While it is known that specific RNA-binding proteins (RBPs) play a role in EV-RNA sorting, the quantitative contribution of competing RBP-RNA interactions and the predictability of RNA-sorting outcomes are poorly understood. Here, we show that a core of hnRNPs compete for the binding to the heterogeneous EV-RNA in vitro. Given prioritized interactions with purine-rich RNA motifs, we set up a pharmacological screen platform to find inhibitors of protein-RNA interactions. Our results suggest that selected small molecules can interfere with EV-RNA quality, altering the distribution of specific miRNA cargoes and associating with a discriminant NF-kB activation in EV-recipient cells. This work highlights the role of RBP-RNA interactions in influencing the EV-RNA quality and paracrine functions.