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Extracellular Vesicle RNA: MicroRNA, Y RNA and tRNA Fragments

Por Benn Bluestein-Veyra, M.Sc. en Química Orgánica · Actualizado el 20 de octubre de 2026

Extracellular vesicle RNA is a mixture of small noncoding RNAs in which transfer RNAs, Y RNAs and ribosomal RNA fragments account for most sequencing reads and microRNA is a minor share.^[1][2] Much of the microRNA found outside cells travels in protein complexes and lipoproteins that co-isolate with vesicles.^[3][4]

This page covers the discovery of vesicle RNA, its biotype composition, Y RNA and its binding protein Ro60, microRNA copy number per vesicle, the non-vesicular carriers of extracellular RNA, the NIH exRNA Atlas and the methods that measure RNA cargo.

Exosomes were shown to carry messenger RNA and microRNA in 2007

Valadi and colleagues reported in 2007 that exosomes from mouse and human mast cells contain RNA. Microarrays detected messenger RNA from about 1,300 genes, many of them absent from the cytoplasm of the donor cell, and the exosome messenger RNAs were translated in vitro.^[5]

The same study found small RNAs, including microRNAs, in exosome RNA. When mouse exosomal RNA was transferred to human mast cells, new mouse proteins appeared in the recipient cells, and the authors named the cargo exosomal shuttle RNA.^[5]

This work opened the field of extracellular RNA. The NIH later funded the Extracellular RNA Communication program through its Common Fund to study how RNA is exported from cells and what roles it plays in communication between cells.^[6][7]

Sequencing shows that small noncoding RNAs dominate extracellular vesicle RNA

Shurtleff and colleagues sequenced RNA from HEK293T cell vesicles purified by flotation gradient, using TGIRT sequencing, which relies on a thermostable group II intron enzyme. Small noncoding RNAs made up 62 percent of reads, ribosomal RNA 27 percent and protein-coding transcripts 6.6 percent (Fig. 1).^[1]

Transfer RNAs alone accounted for 50 percent of all reads and Y RNAs for 8.7 percent, while microRNAs made up less than 0.5 percent. Exosomes captured with antibodies to CD63 gave a similar profile, with transfer RNAs at 66 percent of reads.^[1]

Earlier deep sequencing of RNA released by immune cells, by Nolte-’t Hoen and colleagues, found vault RNA, Y RNA and specific transfer RNA sequences among the most abundant small RNAs. Many were enriched relative to cellular RNA, suggesting that cells select particular RNAs for release.^[8]

Transfer RNAs and their fragments form the largest class

In HEK293T exosomes, about 35 percent of transfer RNA reads spanned the full-length mature molecule of about 75 nucleotides, and the relative abundance of each transfer RNA matched that of the parent cell.^[1] Shorter reads belong to the family now called tRNA-derived fragments.^[9]

Baglio and colleagues sequenced exosomes from human adipose and bone marrow mesenchymal stem cells. Transfer RNAs made up more than half of small RNA in adipose-derived exosomes and 23 to 35 percent in bone marrow exosomes, and tRNA halves in particular were preferentially released.^[2]

MicroRNA is a minority biotype with a selected profile

In the same cells, microRNA was 19 to 49 percent of cellular small RNA and 2 to 5 percent of the exosome small RNA repertoire. A defined set of microRNAs was overrepresented in exosomes compared with the parent cells, and several highly expressed cellular microRNAs were held back from exosomal sorting.^[2]

Driedonks and colleagues analyzed highly pure vesicles from primary dendritic cells. Activating and suppressing immune stimuli changed the microRNA, snoRNA and Y RNA content of the vesicles, while transfer RNA and snRNA levels changed much less.^[10]

Composition also varies between body fluids. Godoy and colleagues sequenced small RNA in 12 human biofluids and found that microRNAs and tRNA-derived RNAs held most mapped reads, with the ratio of microRNA to tRNA-derived reads ranging from 72 in plasma to 0.004 in bile.^[11]

Y RNAs bind the ring-shaped protein Ro60

Y RNAs are a family of conserved noncoding RNAs whose best-known partner is the RNA-binding protein Ro60, encoded in humans by the RO60 gene.^[12][13] Boccitto and Wolin describe Ro60 as a ring-shaped protein that binds Y RNAs on its outer surface and misfolded noncoding RNAs in its central cavity.^[13]

Stein and colleagues solved the structure of Xenopus Ro60 with RNA in 2005 (Fig. 2). The protein combines a von Willebrand factor A domain with a doughnut-shaped ring of HEAT repeats; a Y RNA helix binds the outer surface of the ring and single-stranded RNA enters the central hole.^[14]

Y RNAs and their fragments are among the most abundant extracellular RNAs. Godoy and colleagues found Y RNA fragments in every biofluid they sequenced, at more than 10 percent of reads in plasma, serum and cerebrospinal fluid.^[11]

Driedonks and Nolte-’t Hoen review how extracellular Y RNA is found both in vesicles and in ribonucleoprotein complexes, and they list the RNA-binding proteins known to bind it. Assigning a Y RNA signal to one of these carriers calls for separation of the two fractions.^[15]

Quantitative studies measure microRNA copy number per vesicle

Chevillet and colleagues counted both vesicles and microRNA molecules in exosome preparations from plasma, seminal fluid, dendritic cells, mast cells and a cultured cell line. Across six sources the mean was 0.00825 molecules of a given microRNA per exosome, even for the most abundant microRNAs.^[16]

At that stoichiometry, more than 100 exosomes would need to be examined to observe one copy of an abundant microRNA if copies were spread evenly. The authors proposed revised models to reconcile vesicle-mediated microRNA communication with these numbers.^[16]

The ISEV position paper on vesicle RNA, led by Mateescu, reviews the strategies used to isolate and characterize the small amounts of RNA in vesicles. It sets out the possibilities and limits of each strategy and calls for careful interpretation of the data they produce.^[17]

Argonaute 2 complexes carry much of the microRNA in plasma

Arroyo and colleagues fractionated human plasma and serum by differential centrifugation and size-exclusion chromatography. Most circulating microRNAs cofractionated with protein complexes, and protease digestion of plasma left them open to plasma RNases.^[3]

Argonaute 2, the effector protein of microRNA-guided silencing, eluted with plasma microRNAs, and immunoprecipitating it recovered the non-vesicular microRNAs. A minority of specific microRNAs associated mainly with vesicles, revealing two circulating populations.^[3]

Turchinovich and colleagues reached the same picture independently. Most microRNA in plasma and culture medium passed through 0.22 µm filters, stayed in the supernatant after ultracentrifugation at 110,000 g and co-immunoprecipitated with the 96 kDa Argonaute 2 protein.^[18]

The Argonaute 2 structure explains the stability of the complex

Argonaute 2 is encoded by the human AGO2 gene and is the catalytic component of the RNA-induced silencing complex.^[19] Schirle and MacRae solved its 2.3 Å structure, a bilobed molecule with a central cleft that holds guide RNA nucleotides 2 to 6 in a conformation ready to pair with target messenger RNA.^[20]

Elkayam and colleagues solved Argonaute 2 bound to the microRNA miR-20a (Fig. 3). The microRNA is anchored at both ends by the MID and PAZ domains, and its binding locks the otherwise flexible protein into a stable conformation, consistent with the persistence of these complexes outside cells.^[18][21]

Jeppesen and colleagues used high-resolution density gradients and direct immunoaffinity capture to reassess exosome composition. RNA and RNA-binding proteins partitioned differently between exosomes and non-vesicle fractions, and Argonaute 1 to 4 were absent from the captured exosomes.^[22]

Lipoproteins, exomeres and supermeres also carry extracellular RNA

Vickers and colleagues showed in 2011 that high-density lipoprotein (HDL) transports endogenous microRNAs in plasma (Fig. 4). Export of microRNAs from cells to HDL was regulated by neutral sphingomyelinase, and uptake of HDL-bound microRNAs by recipient cells depended on scavenger receptor class B type I.^[4]

Zhang and colleagues used asymmetric-flow field-flow fractionation to resolve two exosome subsets of 60 to 80 nm and 90 to 120 nm, plus non-membranous nanoparticles of about 35 nm that they named exomeres. Each class carried its own protein, lipid, DNA and RNA profile.^[23]

A later study from the Coffey laboratory, led by Zhang, described supermeres, nanoparticles morphologically distinct from exomeres. Their protein and RNA composition differed from that of small vesicles and exomeres, and supermeres held the majority of extracellular RNA.^[24]

Extracellular RNA can also change after release. Tosar and colleagues found ribosomes and full-length transfer RNAs in conditioned medium from several mammalian cell lines, and their fragmentation outside the cell generated small RNAs that are highly resistant to degradation.^[25]

The non-vesicular fraction is biased toward specific tRNA-derived fragments that form RNase-protecting dimers, and Tosar and Cayota review which fragments dominate each extracellular fraction. Extracellular abundance therefore reflects both secretion and stability.^[9][25]

The exRNA Atlas maps RNA cargo types across human biofluids

The Extracellular RNA Communication Consortium (ERCC), funded by the NIH Common Fund, set out to build reference profiles of extracellular RNA from healthy individuals, define how it is made and taken up, and share protocols, data and tools through the exRNA Atlas.^[6][7]

Das and colleagues summarized the first phase of the consortium, including collaborative projects on methods for vesicle and extracellular RNA isolation and on tools for sharing and analyzing profiling data.^[26]

Murillo and colleagues analyzed 5,309 profiles from 19 studies in the exRNA Atlas by computational deconvolution. The model identified six extracellular RNA cargo types, each detectable in several of serum, plasma, cerebrospinal fluid, saliva and urine, and five of them associated with vesicular, lipoprotein or ribonucleoprotein carriers.^[27]

Srinivasan and colleagues compared 10 isolation methods across 5 biofluids. Methods differed markedly in the complexity and reproducibility of small RNA profiles and in how much they captured from vesicle, ribonucleoprotein and HDL carriers, measured by carrier-specific microRNA signatures.^[28]

Measuring extracellular vesicle RNA combines separation, protection and sequencing

Each published composition depends on how vesicles were separated from co-isolating carriers. MISEV2023 sets out the approaches for separating vesicles from non-vesicular extracellular particles and for characterizing the resulting preparations.^[17][29]

• Density gradient flotation or high-resolution density fractionation, which separates vesicles from protein complexes.^[1][22]
• Size-exclusion chromatography, which resolves vesicles, Argonaute 2 complexes and smaller particles by elution volume.^[3][25]
• Nuclease protection, which assigns RNA to the vesicle lumen when it resists RNase until a detergent opens the membrane.^[1]
• Small RNA sequencing, where the isolation method and the library chemistry both shape the biotype shares a study reports.^[1][28]

The nuclease protection assay reads location from a series of enzyme and detergent steps (Fig. 5). In HEK293T vesicles, transfer RNA and other small noncoding RNAs resisted RNase and protease and were degraded only with detergent, which placed them inside the vesicles.^[1]

The same experiments traced many reads mapping to protein-coding genes to DNA on the vesicle surface, and the authors measured messenger RNA in DNase-digested samples.^[1] For non-vesicular samples, adding RNase inhibitors revealed ribosomes and full-length transfer RNAs in the extracellular fraction.^[25]

Where to go from here

The exomeres and supermeres reference covers the nanoparticles that travel with vesicles, and the cargo sorting reference covers how RNA-binding proteins select RNAs for release. The uptake reference follows vesicles to recipient cells.

The quality reference on MISEV2023 explains what a vesicle preparation can be documented against. Verified practices can request lot-specific characterization data with the certificate of analysis, and our team can explain how a preparation was separated and measured.