Interfering with hnRNPA2B1:ExoRNA interaction to challenge the quality of secreted extracellular vesicles

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease. In ALS, RNA-binding proteins (RBPs) accumulate and/or misfold and eventually associate with stress granules, leading to proteotoxic damage and RNA toxicity, ultimately resulting in neurodegeneration. Extracellular vesicles (EVs) are released by most cells to the extracellular environment and are involved in cell-to-cell communication. In ALS, EVs have been proposed as a vehicle through which “meaningful signals” are delivered prompting the propagation of the disease. MicroRNA biomarkers are recently emerging as EVs cargo. To this extent, the activity of RBPs needs to be addressed in view of the enrichment of selected transcripts into EVs (EV-RNA). Among RBPs, the ALS-linked hnRNPA2B1 was discovered as a key player in the sorting of selected miRNA in exosomes (or small EVs). For these reasons, we proposed hnRNPA2B1 and its interaction with RNA as an interesting target for drug screening in ALS. To study the mechanism of EV-RNA modulation, we optimized a proteomic approach and measured the EV-RNA upon modulation of hnRNPA2B1 in NSC-34 cells, finding that the protein effectively contributes to this regulation. Producing a human full-length hnRNPA2B1 protein and an RNA probe harboring the EXOmotif, previously known to be enriched in exosomal RNA, we performed a high-throughput drug screening with the aim to identify drugs able to interfere with hnRNPA2B1-RNA interaction. We screened a library of 2000 compounds, containing FDA-approved and natural scaffolds, and we identified 21 hits. We performed a counter screening using AlphaScreen and REMSA orthogonal techniques and we identified six active compounds able to interfere with protein:RNA interaction; RNA pull-down assay confirmed the compounds effects on endogenous Hnrnpa2b1 in NSC-34. To assess the interference with EV-RNA quality, we looked at miR-221-3p as a readout to measure vesicular hnRNP2AB1-regulated miRNAs. miR-221-3p levels resulted to be significant reduced, upon treatment, in NSC-34 and in NPC-derived motor neurons EVs. Therefore, we started some pilot experiments looking at the effects of these modulated EVs on recipient cells. miR-221-3p deprived-EVs, upon compound treatments, associated with hnRNPA2B1-mediated NF-kB activation in recipient cells. Overall, we identified powerful compounds able to affect EV-RNA quality. This demonstrates the possibility to biochemically interfere with the EV-RNA cargo without altering the global particle release.