Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Kim, Hyun Ji; Rames, Matthew J.; Roskams-Hieter, Breeshey; Briand, Joséphine; Doe, Aaron; Estabrook, Joseph; Wagner, Josiah Tad; Demir, Emek; Mills, Gordon B.; Ngo, Thuy Minh

doi:10.1101/2022.09.22.509102

Cited by 2 publications

(1 citation statement)

References 57 publications

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“…Coding and non-coding RNAs can be detected in secreted EVs 13,14 . Competing RBPs could significantly contribute to selecting EV transcripts, including, for example, miRNAs 15 , demonstrating that selective packaging can occur at an intracellular level for a desired secretion.…”

Section: Introductionmentioning

confidence: 99%

Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles

Corsi,

Peroni,

Belli

et al. 2024

Preprint

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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-κB 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-RNAin 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.

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Section: Introductionmentioning

confidence: 99%

Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles

Corsi,

Peroni,

Belli

et al. 2024

Preprint

View full text Add to dashboard Cite

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Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Kim,

Rames,

Goncalves

et al. 2023

Commun Biol

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Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluids and their association with cancer remain poorly understood. Here, we performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of large and medium particles from other soluble plasma protein fractions. We developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from particle and protein enriched subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in cf-mRNA amount carried in each plasma fraction. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cancer-associated cf-mRNA in each particle and protein enriched subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EV subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.

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Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Cited by 2 publications

References 57 publications

Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles

Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles

Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

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