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SMALL MOLECULE INHIBITORS OF HNRNPA2B1-RNA INTERACTIONS REVEAL A PREDICTABLE SORTING OF RNA SUBSETS INTO EXTRACELLULAR VESICLES

ABSTRACT: Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and non-coding RNAs are widely detected as EV cargo, and RNAbinding 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 and used cells expressing a recombinant hnRNPA2B1 to normalize competitive interactions. Interestingly, we prioritized heterogeneous nuclear ribonucleoproteins in networks including RAB proteins and recognizing purine-rich RNA sequences representing a subset of previously identified EXO motifs. A screening campaign using a full-length human hnRNPA2B1 protein and a synthetic purine-rich RNA probe 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-kB in EV 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Romina Belli

LAB HEAD: Vito G. D'Agostino

PROVIDER: PXD052658 | Pride | 2025-02-24

REPOSITORIES: pride

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Project description:We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, pro-angiogenic, anti-fibrotic and proliferative effects on target cells. hAFS can be also isolated from III trimester clinical waste samples during C-section scheduled procedures (perinatal hAFS), thus offering an easily accessible alternative source. Nonetheless, little is known about their paracrine profile. Here we provide a detailed characterization of the hAFS total secretome (i.e. whole of the soluble paracrine factors released by the cells in their conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it as from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. Their secretome formulations were analyzed in terms of protein and chemokine/cytokine content, with the EV cargo further investigated by RNA sequencing. While the appearance of fetal and perinatal hAFS, along with their corresponding secretome formulations was overlapping, the profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. While both cell sources provided paracrine formulations enriched with neurotrophic, immunomodulatory and endothelial stimulating factors, the more immature fetal hAFS secretome was defined by a more pronounced pro-angiogenic, pro-resolving and anti-aging profile over the perinatal one, while the corresponfing EV…. Moreover, small RNA profiling showed a relevant enrichment of microRNA in both the fetal and perinatal hAFS-EV cargo, with a steadily expressed pro-resolving miRNA core as reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

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SMALL MOLECULE INHIBITORS OF HNRNPA2B1-RNA INTERACTIONS REVEAL A PREDICTABLE SORTING OF RNA SUBSETS INTO EXTRACELLULAR VESICLES

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