Extracellular vesicles (EVs) are heterogeneous membranous particles released from the cells through different biogenetic and secretory mechanisms. We now conceive EVs as shuttles mediating cellular communication, carrying a variety of molecules resulting from intracellular homeostatic mechanisms. The RNA is a widely detected cargo and, impressively, a recognized functional intermediate that elects EVs as modulators of cancer cell phenotypes, determinants of disease spreading, cell surrogates in regenerative medicine, and a source for non‐invasive molecular diagnostics. The mechanistic elucidation of the intracellular events responsible for the engagement of RNA into EVs will significantly improve the comprehension and possibly the prediction of EV “quality” in association with cell physiology. Interestingly, the application of multidisciplinary approaches, including biochemical as well as cell‐based and computational strategies, is increasingly revealing an active RNA‐packaging process implicating RNA‐binding proteins (RBPs) in the sorting of coding and non‐coding RNAs. In this review, we provide a comprehensive view of RBPs recently emerging as part of the EV biology, considering the scenarios where: (i) individual RBPs were detected in EVs along with their RNA substrates, (ii) RBPs were detected in EVs with inferred RNA targets, and (iii) EV‐transcripts were found to harbour sequence motifs mirroring the activity of RBPs. Proteins so far identified are members of the hnRNP family (hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPH1, hnRNPK, and hnRNPQ), as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, TDP‐43, MVP, LIN28, SRP9/14, QKI, and TERT. We describe the RBPs based on protein domain features, current knowledge on the association with human diseases, recognition of RNA consensus motifs, and the need to clarify the functional significance in different cellular contexts. We also summarize data on previously identified RBP inhibitor small molecules that could also be introduced in EV research as potential modulators of vesicular RNA sorting.

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Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC.

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 μM and a good selectivity over the paralog BAZ2B bromodomain.

The bromodomains of BAZ2A and BAZ2B (bromodomain adjacent to zinc finger domain proteins 2) are among the most hard to drug of the 61 human bromodomains. While little is known about the role of BAZ2B, there is strong evidence for the opportunity of targeting BAZ2A in various cancers. Here, a benzimidazole−triazole fragment that binds to the BAZ2A acetyl lysine pocket was identified by a molecular docking campaign and validated by competitive binding assays and X-ray crystallography. Another ligand was observed in close proximity by soaking experiments using the BAZ2A bromodomain preincubated with the benzimidazole−triazole fragment. The crystal structure of BAZ2A with the two ligands was employed to design a few benzimidazole−triazole derivatives with increased affinity. We also present the engineering of a BAZ2A bromodomain mutant for consistent, high-resolution crystallographic studies.