Diversity and heterogeneity of extracellular RNA in human plasma

Galvanin, Adeline; Dostert, Gabriel; Ayadi, Lilia; Marchand, Virginie; Velot, Émilie; Motorin, Yuri

doi:10.1016/j.biochi.2019.05.011

Cited by 30 publications

(26 citation statements)

References 89 publications

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“…GAA purity by deep sequencing was performed by mild RNA fragmentation under strong alkaline conditions (5 min, 96°C, pH 9.2). Resulting fragments were 3′-dephosphorylated by Antarctic phosphatase (New England Biolabs, Frankfurt, Germany) and 5′-phosphorylated by PNK/ATP treatment 52 . Library preparation was done using NEBNext ® Multiplex Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer's instructions.…”

Section: Sequencing Analysis Of Trna Phementioning

confidence: 99%

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

et al. 2021

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Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

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Section: Sequencing Analysis Of Trna Phementioning

confidence: 99%

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

et al. 2021

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“…Importantly, several RNAs are stable and longly detectable in human bloodstream (Umu et al 2018; Galvanin et al 2019), indeed some of these circulating and stable RNAs are already used to challenge the effectiveness of patient treatment (Guo et al 2015). These observations highlight the usefulness of companion diagnostics in clinical settings that would become solid diagnostic and prognostic markers in the future.…”

Section: Discussionmentioning

confidence: 99%

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance

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et al. 2021

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FTSJ1 is a phyllogenetically conserved human 2-O-methyltransferase (Nm-MTase) which modifies position 32 as well as the wobble position 34 in the AntiCodon Loop (ACL) of specific tRNAs: tRNAPhe(GAA), tRNATrp(CCA) and tRNALeu(UAA). FTSJ1 loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently in cancers. However, the exact molecular mechanisms underlying FTSJ1-related pathogenesis are unknown and a potential extended variety of FTSJ1 tRNA targets has not been fully addressed yet. We performed unbiased and comprehensive RiboMethSeq analysis of the Nm profiles for human tRNA population extracted from cells derived from NSXLID patients blood bearing various characterized loss of function mutations in FTSJ1. In addition, we reported a novel FTSJ1 pathogenic variant from a NSXLID patient bearing a de novo mutation in the FTSJ1 gene. Some of the newly identified FTSJ1 tRNA targets are also conserved in Drosophila as shown by our previous study on the fly homologues Trm7_32 and Trm7_34, whose loss affects small RNA silencing pathways. In the current study, we reveal a conserved deregulation in both the miRNA and mRNA populations when FTSJ1 function is compromised. In addition, a cross-analysing between deregulated miRNA and mRNA obtained in FTSJ1 mutants highlighted upregulation of miR10a-5p which has the capacity to silence the SPARC gene mRNA, downregulated in FTSJ1 mutant cells. This suggests that FTSJ1 loss may influence gene expression deregulation by modulation of miRNA silencing. A gene-ontology (GO) enrichment analysis of the deregulated mRNAs primarily matched to brain morphogenesis terms, followed by metabolism and translation related genes. In parallel, the deregulated miRNAs are mostly known for their implication in brain functions and cancers. Based on these results, we suggest that miRNA silencing variations may play a role in the pathological mechanisms of FTSJ1-dependent NSXLID. Finally, our results highlight miR-181a-5p as a potential companion diagnostic test in clinical settings for FTSJ1-related intellectual disability.

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“…A large proportion of circulating RNAs, including miRNAs, are contributed by the microbiome. In some EV preparations, 75% of the total RNA reads originate exogenously (Wang et al, 2012 ; Fritz et al, 2016 ; Galvanin et al, 2019 ). Tulkens et al outlined a newly-developed combinatorial strategy including ultrafiltration, chromatography, and density gradient ultracentrifugation to fractionate bacterial- and host-derived EVs and other contaminating particles (Tulkens et al, 2020 ).…”

Section: Extracellular Vesicle Physiology and Biomedical Relevancementioning

confidence: 99%

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

2020

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Cells release nanometer-scale, lipid bilayer-enclosed biomolecular packages (extracellular vesicles; EVs) into their surrounding environment. EVs are hypothesized to be intercellular communication agents that regulate physiological states by transporting biomolecules between near and distant cells. The research community has consistently advocated for the importance of RNA contents in EVs by demonstrating that: (1) EV-related RNA contents can be detected in a liquid biopsy, (2) disease states significantly alter EV-related RNA contents, and (3) sensitive and specific liquid biopsies can be implemented in precision medicine settings by measuring EV-derived RNA contents. Furthermore, EVs have medical potential beyond diagnostics. Both natural and engineered EVs are being investigated for therapeutic applications such as regenerative medicine and as drug delivery agents. This review focuses specifically on EV characterization, analysis of their RNA content, and their functional implications. The NIH extracellular RNA communication (ERC) program has catapulted human EV research from an RNA profiling standpoint by standardizing the pipeline for working with EV transcriptomics data, and creating a centralized database for the scientific community. There are currently thousands of RNA-sequencing profiles hosted on the Extracellular RNA Atlas alone (Murillo et al., 2019 ), encompassing a variety of human biofluid types and health conditions. While a number of significant discoveries have been made through these studies individually, integrative analyses of these data have thus far been limited. A primary focus of the ERC program over the next five years is to bring higher resolution tools to the EV research community so that investigators can isolate and analyze EV sub-populations, and ultimately single EVs sourced from discrete cell types, tissues, and complex biofluids. Higher resolution techniques will be essential for evaluating the roles of circulating EVs at a level which impacts clinical decision making. We expect that advances in microfluidic technologies will drive near-term innovation and discoveries about the diverse RNA contents of EVs. Long-term translation of EV-based RNA profiling into a mainstay medical diagnostic tool will depend upon identifying robust patterns of circulating genetic material that correlate with a change in health status.

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Diversity and heterogeneity of extracellular RNA in human plasma

Cited by 30 publications

References 89 publications

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

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