The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles

Liu, Daniel; Yang, Qi Zhi Clayton; Asim, Mohammad; Krell, Jonathan; Frampton, Adam E.

doi:10.3390/ijms23073692

Cited by 12 publications

(11 citation statements)

References 89 publications

(123 reference statements)

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“…Our data reveal that the gene number in a single‐EV varied from 6 to 148; this differs from a previous report that one mRNA copy or mRNA fragment can be found in ≈10 EVs. [ 19a ] Consistent with previous studies, [ 11,20 ] we found that rRNA and mitochondrial RNA are abundant in K562‐EV and MSC‐EV. Characterizing the expression of mitochondrial genes in EVs helps to explain the release and transport of extracellular mitochondria.…”

Section: Discussionsupporting

confidence: 92%

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Luo

Chen

Qiu³

et al. 2022

Small Methods

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body fluids. EVs are highly heterogenous in size, ranging from 50 to 1000 nm in diameter. [1] Furthermore, they play key roles in the cell microenvironment and intercellular communication by transferring biological molecules including proteins, lipids, and nucleic acids. [2] Because of the relatively stable state in body fluids, EVs are recognized as potential noninvasive biomarkers in disease diagnostics and mediators in drug delivery. [3] EVs carry the characteristics of their parental cells; therefore, they are highly heterogeneous in molecular cargos. [4] Thus, EVs demonstrate a marked diversity, even in homogeneous or monoclonal cell populations. [5] The different molecular cargos in EVs are related to their function and effectiveness as disease biomarkers. Therefore, it is important to investigate the molecular cargos and their heterogeneity at the single-EV level.Traditional analytical methods to examine the molecular cargos of EVs, such as western blotting, polymerase chain reaction (PCR), and RNA-seq, usually rely on bulk assays to determine the overall molecular content from an entire EV population. The molecular cargo of an individual EV and the heterogeneity within an EV population is lost in bulk-level analyses. Recently, several new analytical methods, such as total internal reflection fluorescence microscopy (TIRFM), [6] digital PCR, [7] microarraybased chip, [8] and high-sensitivity flow cytometry combined with qPCR, [9] have been used to quantify a specific miRNA or mRNA in an single-EV or small identical subpopulations. However, a high-throughput analysis for the RNA profile of a single-EV is still lacking. Therefore, even though many studies have investigated the RNA cargos in bulk EVs, we still do not know how many genes or RNAs there are in a single-EV. This is a basic issue that needs to be addressed. Investigations of transcriptome for a single-EV are of great value to better define EV features, EV subpopulations, and clinical applications.The development of single-cell RNA sequencing (scRNA-seq) technologies, such as 10x Genomics Chromium, enable the detection of gene expression at the single-cell level, as well as the detection of cell heterogeneity and cell types. [10] It has been reported that EV populations are more heterogeneous than their parental cells. [11] Therefore, it is very important to build an experimental and analytical protocol to perform single-EV sequencing to reveal the transcriptome features of individual EVs.Although many studies have investigated functional molecules in extracellular vesicles (EVs), the exact number of ribonucleic acid molecules in a single-EV is unknown. Therefore, it is critical to explore the transcriptomic features and heterogeneity at the level of a single-EV. Here, using the 10x Genomics platform, the RNA cargos are profiled in single EVs derived from human K562 and mesenchymal stem cells. The key steps are labeling intact EVs using calcein-AM, detecting the EV concentration via flow cytometry, and using the CB2 algorithm with adaptive thres...

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Section: Discussionsupporting

confidence: 92%

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Luo

Chen

Qiu³

et al. 2022

Small Methods

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“…The clinical significance of tRNAs and tRFs in EVs has recently been reviewed by Liu et al (2022) and Weng et al (2022).…”

Section: Discussionmentioning

confidence: 99%

“…This highlights the need for standardised nomenclature for the different tRFs to enable inter-study comparisons and metanalyses. The clinical significance of tRNAs and tRFs in EVs has recently been reviewed by Liu et al (2022) and Weng et al (2022).…”

Section: Discussionmentioning

confidence: 99%

Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages

Pantazi

Clements

Abrahams

et al. 2022

Preprint

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Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely piRNAs, snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment.

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“…Studies performed mainly in eukaryotes have shown that transfer RNAs (tRNAs) are an emerging source of functional sRNAs in the tRNA-derived fragments (tRFs) they generate (Kumar et al, 2016;Kuscu et al, 2018). Eukaryotic tRFs are involved in several functions ranging from translation inhibition to repression (Keam and Hutvagner, 2015;Kazimierczyk et al, 2022;Liu et al, 2022). In bacteria, little is known about the biogenesis, characteristics and cell-autonomous function of tRFs [for reviews, see ref.…”

Section: Introductionmentioning

confidence: 99%

RNA Sequencing Unveils Very Small RNAs With Potential Regulatory Functions in Bacteria

Diallo

Lalaouna

et al. 2022

Front. Mol. Biosci.

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RNA sequencing (RNA-seq) is the gold standard for the discovery of small non-coding RNAs. Following a long-standing approach, reads shorter than 16 nucleotides (nt) are removed from the small RNA sequencing libraries or datasets. The serendipitous discovery of an eukaryotic 12 nt-long RNA species capable of modulating the microRNA from which they derive prompted us to challenge this dogma and, by expanding the window of RNA sizes down to 8 nt, to confirm the existence of functional very small RNAs (vsRNAs <16 nt). Here we report the detailed profiling of vsRNAs in Escherichia coli, E. coli-derived outer membrane vesicles (OMVs) and five other bacterial strains (Pseudomonas aeruginosa PA7, P. aeruginosa PAO1, Salmonella enterica serovar Typhimurium 14028S, Legionella pneumophila JR32 Philadelphia-1 and Staphylococcus aureus HG001). vsRNAs of 8–15 nt in length [RNAs (8-15 nt)] were found to be more abundant than RNAs of 16–30 nt in length [RNAs (16–30 nt)]. vsRNA biotypes were distinct and varied within and across bacterial species and accounted for one third of reads identified in the 8–30 nt window. The tRNA-derived fragments (tRFs) have appeared as a major biotype among the vsRNAs, notably Ile-tRF and Ala-tRF, and were selectively loaded in OMVs. tRF-derived vsRNAs appear to be thermodynamically stable with at least 2 G-C basepairs and stem-loop structure. The analyzed tRF-derived vsRNAs are predicted to target several human host mRNAs with diverse functions. Bacterial vsRNAs and OMV-derived vsRNAs could be novel players likely modulating the intricate relationship between pathogens and their hosts.

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The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles

Cited by 12 publications

References 89 publications

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages

RNA Sequencing Unveils Very Small RNAs With Potential Regulatory Functions in Bacteria

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