exRNA-eCLIP intersection analysis reveals a map of extracellular RNA binding proteins and associated RNAs across major human biofluids and carriers

LaPlante, Emily L.; Sturchler, Alessandra; Fullem, Robert; Chen, David; Starner, Anne C.; Esquivel, Emmanuel; Alsop, Eric; Jackson, Andrew; Ghiran, Ionita; Pereira, Getulio; Rozowsky, Joel; Chang, Justin; Gerstein, Mark; Alexander, Roger P.; Roth, Matthew E.; Franklin, Jeffrey L.; Coffey, Robert J.; Raffaı̈, Robert L.; Mansuy, Isabelle M.; Stavrakis, Stavros; deMello, Andrew J.; Laurent, Louise C.; Wang, Yiting; Tsai, Chia‐Feng; Liu, Tao; Jones, Jennifer; Keuren‐Jensen, Kendall Van; Nostrand, Eric Van; Mateescu, Bogdan; Milosavljevic, Aleksandar

doi:10.1016/j.xgen.2023.100303

Cited by 8 publications

(4 citation statements)

References 32 publications

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“…However, several RNA species are stable in the extracellular space even when not associated with EVs. These resilient extracellular RNAs include ribonucleoprotein particles (RNPs) such as Ago2/miRNA complexes (Turchinovich et al 2011; Arroyo et al 2011; Geekiyanage et al 2020), and protein-protected RNA fragments (LaPlante et al 2023) derived from the ribosome (Tosar et al 2020, 2022; Costa et al 2023), U2 snRNPs (Tosar et al 2022), and possibly Xist RNPs (Dou et al 2024). Additionally, a population of intrinsically stable nicked tRNAs circulates in human biofluids, probably unprotected or naked (Costa et al 2023).…”

Section: Discussionmentioning

confidence: 99%

Ribonuclease activity undermines immune sensing of naked extracellular RNA

Castellano,

Blanco,

Calzi

et al. 2024

Preprint

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The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

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Section: Discussionmentioning

confidence: 99%

Ribonuclease activity undermines immune sensing of naked extracellular RNA

Castellano,

Blanco,

Calzi

et al. 2024

Preprint

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“…Dr. Aleksandar Milosaviljevic (Baylor College of Medicine) shared his experience from the exRNA Consortium and questioned whether it is advisable to focus only on EVs or to broaden the scope of studies to include other particles, like lipoproteins and ribonucleoproteins, as these Non-Vesicular Extracellular Particles (NVEPs) share protein and exRNA cargo and may not always be separated experimentally. One example is provided by the extracellular RNA-Binding Proteins (exRBPs) and associated exRNA, which may be found in both EVs and NVEPs across human biofluids, as evidenced by the recent intersection analysis of eCLIP and exRNA Atlas data (LaPlante et al, 2023). Yari Ciani (University of Trento, Italy), Shiro Suetsugu (Nara Institute of Science and Technology, Japan), and Alice Gualerzi (IRCCS Fondazione Don Carlo Gnocchi, Italy) described the application of three different omics approaches (transcriptomics, proteomics, and lipidomics) to address their biological questions with biomolecular, spectrometric and spectroscopic methods, respectively.…”

Section:  Session  Ev Omics Data Analysis: Which Topics Require Th...mentioning

confidence: 99%

International Society for Extracellular Vesicles workshop. QuantitatEVs: Multiscale analyses, from bulk to single extracellular vesicle

Basso,

Gori,

Nardella

et al. 2024

J of Extracellular Bio

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The “QuantitatEVs: multiscale analyses, from bulk to single vesicle” workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV‐associated molecular components and EV biophysical features, which are considered the core of EV‐associated biomarker discovery and validation for their clinical translation. The in‐person‐only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with “Next Generation EVs,” a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

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“…However, the precision of the method mentioned above highly depends on the quality of the original training samples, which ideally should be obtained from the cleanest possible media with well-documented identities. In their recent report, LaPlante et al (2023) proposed an integrative analysis of eCLIP data, which implied the identification of 150 RBPs binding sites in more than 6,000 human EV samples. Specifically, 34 RBPs were indeed detected in blood plasma and conditioned media.…”

Section: Transcriptome Of Extracellular Vesiclesmentioning

confidence: 99%

“…Specifically, 34 RBPs were indeed detected in blood plasma and conditioned media. Remarkably, the deconvolution method described above enabled attributing RBPs to certain exRNA carriers ( LaPlante et al, 2023 ).…”

Section: Transcriptome Of Extracellular Vesiclesmentioning

confidence: 99%

Challenges in characterization of transcriptomes of extracellular vesicles and non-vesicular extracellular RNA carriers

Makarova,

Maltseva,

Tonevitsky

2023

Front. Mol. Biosci.

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Since its original discovery over a decade ago, extracellular RNA (exRNA) has been found in all biological fluids. Furthermore, extracellular microRNA has been shown to be involved in communication between various cell types. Importantly, the exRNA is protected from RNases degradation by certain carriers including membrane vesicles and non-vesicular protein nanoparticles. Each type of carrier has its unique exRNA profile, which may vary depending on cell type and physiological conditions. To clarify putative mechanisms of intercellular communication mediated by exRNA, the RNA profile of each carrier has to be characterized. While current methods of biofluids fractionation are continuously improving, they fail to completely separate exRNA carriers. Likewise, most popular library preparation approaches for RNA sequencing do not allow obtaining exhaustive and unbiased data on exRNA transcriptome. In this mini review we discuss ongoing progress in the field of exRNA, with the focus on exRNA carriers, analyze the key methodological challenges and provide recommendations on how the latter could be overcome.

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exRNA-eCLIP intersection analysis reveals a map of extracellular RNA binding proteins and associated RNAs across major human biofluids and carriers

Cited by 8 publications

References 32 publications

Ribonuclease activity undermines immune sensing of naked extracellular RNA

Ribonuclease activity undermines immune sensing of naked extracellular RNA

International Society for Extracellular Vesicles workshop. QuantitatEVs: Multiscale analyses, from bulk to single extracellular vesicle

Challenges in characterization of transcriptomes of extracellular vesicles and non-vesicular extracellular RNA carriers

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