Corsi Jessica, Semnani Pouriya Sharbatian, Peroni Daniele, Belli Romina, Morelli Alessia, Lassandro Michelangelo, Sidarovich Viktoryia, Adami Valentina, Valentini Chiara, Cavallerio Paolo, Grosskreutz Julian, Fabbiano Fabrizio, Grossmann Dajana, Hermann Andreas, Tell Gianluca, Basso Manuela, D'Agostino Vito G
Laboratory of Biotechnology and Nanomedicine, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Trento, Italy.
MS Core Facility, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Trento, Italy.
Nucleic Acids Res. 2025 Feb 27;53(5). doi: 10.1093/nar/gkaf176.
Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and noncoding RNAs can be detected as EV cargo, and RNA-binding proteins (RBPs), such as hnRNPA2B1, have been circumstantially implicated in EV-RNA sorting mechanisms. However, the contribution of competitive RBP-RNA interactions responsible for RNA-sorting outcomes is still unclear, especially for predicting the EV-RNA content. We designed a reverse proteomic analysis exploiting the EV-RNA to identify intracellular protein binders in vitro. Using cells expressing a recombinant hnRNPA2B1 to normalize competitive interactions, we prioritized a network of heterogeneous nuclear ribonucleoproteins and purine-rich RNA sequences subsequently validated in secreted EV-RNA through short fluorescent RNA oligos. Then, we designed a GGGAG-enriched RNA probe that efficiently interacted with a full-length human hnRNPA2B1 protein. We exploited the interaction to conduct a pharmacological screening and identify inhibitors of the protein-RNA binding. Small molecules were orthogonally validated through biochemical and cell-based approaches. Selected drugs remarkably impacted secreted EV-RNAs and reduced an RNA-dependent, EV-mediated paracrine activation of NF-kB in recipient cells. These results demonstrate the relevance of post-transcriptional mechanisms for EV-RNA sorting and the possibility of predicting the EV-RNA quality for developing innovative strategies targeting discrete paracrine functions.
细胞外囊泡（EVs）是细胞分泌的膜性颗粒，有助于细胞间通讯。编码和非编码RNA可作为EV的货物被检测到，并且RNA结合蛋白（RBPs），如hnRNPA2B1，已间接参与EV-RNA分选机制。然而，负责RNA分选结果的竞争性RBP-RNA相互作用的贡献仍不清楚，特别是在预测EV-RNA含量方面。我们设计了一种反向蛋白质组学分析方法，利用EV-RNA在体外鉴定细胞内蛋白质结合物。使用表达重组hnRNPA2B1的细胞来标准化竞争性相互作用，我们确定了一个异质性核糖核蛋白和富含嘌呤的RNA序列网络，随后通过短荧光RNA寡核苷酸在分泌的EV-RNA中进行了验证。然后，我们设计了一种富含GGGAG的RNA探针，它能与全长人hnRNPA2B1蛋白有效相互作用。我们利用这种相互作用进行了药理筛选，并鉴定了蛋白质-RNA结合的抑制剂。通过生化和基于细胞的方法对小分子进行了正交验证。所选药物显著影响分泌的EV-RNAs，并降低了受体细胞中RNA依赖性、EV介导的NF-κB旁分泌激活。这些结果证明了转录后机制对EV-RNA分选的相关性，以及预测EV-RNA质量以开发针对离散旁分泌功能的创新策略的可能性。
