Unlocking the
Mysteries of
Extracellular RNA

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

The potential of extracellular vesicle (EV) RNA as biomarkers of disease is increasingly being recognized. Circulating extracellular RNAs can potentially indicate the presence of disease without the need for an invasive biopsy of diseased tissue. A recent study by Yuan et al (Scientific Reports, Jan 2016) performed a systematic analysis of circulating exRNA in plasma obtained from 50 healthy individuals and 142 cancer patients. The authors conducted the largest RNA sequencing study reported to date for profiling circulating extracellular RNA (exRNA) species in order to provide useful insights into their baseline expression level. High-throughput multivariate statistical analysis identified a set of RNA candidates that were associated with age, sex, and cancer type.

The study directly addressed a major challenge in the field of exRNA, namely the lack of a baseline reference to accurately determine RNA abundance. Currently qRT-PCR is the most common method used to quantitate gene expression, but it relies on well-established reference controls for normalization. However, information on reference controls has not been established for exRNA. Control RNAs used for normalization in qRT-PCR experiments of cellular RNA cannot be reliably used for exRNA. Exogenous spike-in RNAs such as those derived from species like C. elegans cannot be used to normalize across biological or pathological states. Here the authors surveyed a pool of almost 200 exRNA profiles to identify a few potential reference candidates for exRNA quantification. Most candidate were miRNAs like miR-99a-5p. The study also provided convincing evidence for the presence in plasma of species of RNA other than miRNA, such as piwiRNA (see figure).

The dataset generated by this study is available in the exRNA Atlas for other researchers to explore. Future studies will be needed to validate these findings; however, with the current study we have taken a big leap towards the goal of determining the biomarker potential of exRNA for human diseases.

RNA species detected in the plasma of 142 cancer patients and 50 healthy controls

RNA species detected in the plasma of 142 cancer patients and 50 healthy controls
Source: Scientific Reports / CC BY 4.0

In previous studies in humans, measurement of extracellular RNAs (exRNAs) have primarily focused on microRNAs (miRNAs) or studied a small handful of subjects. The specific question of how large numbers of exRNAs are expressed in broader, non-diseased populations has remained. To examine this question, several groups from multiple institutions collaborated to measure exRNAs in the blood plasma of participants from the Framingham Heart Study, an observational cohort study based in Framingham, MA. In their recent publication (1), they first analyzed RNA sequencing data from the plasma of 40 individuals and identified over a thousand human exRNAs including miRNAs, piwi-interacting RNA (piRNAs), and small nucleolar RNAs (snoRNAs).

Study Design

Study Design

Although miRNAs have been commonly observed in the circulation and plasma, little is known about the presence of other common varieties of small human RNAs such as piRNAs and snoRNAs, known to be key components of molecular interactions and gene regulation in eukaryotes. Using a targeted RT-qPCR approach in an additional 2,763 individuals, the groups then characterized almost 500 of the most abundant extracellular RNA transcripts. The presence in plasma of many non-microRNA small RNAs was confirmed in this independent cohort. The findings show that diverse classes of circulating non-cellular small RNAs, beyond miRNAs, are consistently present in plasma from multiple human populations. Further work will determine how the presence of these exRNAs in the circulation correlates with the presence and progression of a broad number of human traits and diseases.

1. Freedman JE, Gerstein M, Mick E, Rozowsky J, Levy D, Kitchen R, Das S, Shah R, Danielson K, Beaulieu L, Navarro FCP, Wang Y, Galeev TR, Holman A,, Kwong RY, Murthy V, Tanriverdi SE, Koupenova-Zamor M, Mikalev E, Tanriverdi K. Diverse Human Extracellular RNAs are Widely Detected in Plasma. Nature Communications. Published online 26 April 2016.

Overly active KRAS leads to increased serine phosphorylation of Ago2 downstream of MEK and ERK. Phosphorylated Ago2 associates more with P-bodies than with multivesicular endosomes, which reduces the sorting of Ago2 and miRNAs into exosomes bound for export from the cell.

Source: Cell Reports


miRNA release into extracellular vesicles (EVs) is a mechanism to control the gene expression and cellular phenotypes of neighboring cells. A key question is how specific miRNAs are sorted into EVs. Active sorting of RNAs to extracellular carriers such as EVs likely depends on binding to specific RNA binding proteins. As a key member of the RNA-induced silencing complex (RISC) machinery that directly binds miRNA, Argonaute 2 (Ago2) has been a strong candidate as a miRNA carrier in EVs. However, the presence of Ago2 in EVs has been controversial.

In a new paper, we show that Ago2 is carried in both microvesicles and exosomes. Using isogenic cell lines for mutant oncogenic KRAS, we show that Ago2 sorting to exosomes is specifically down-regulated by KRAS-MEK-ERK signaling at late endosomes. Tests of three candidate miRNAs showed that this mechanism can regulate sorting of miRNAs to exosomes. Overall, these data indicate that Ago2 sorting to exosomes is a regulated event and may control miRNA sorting. Furthermore, previous studies that were performed in the presence of serum or growth factors in the media may have detected little Ago2 in exosomes due to growth factor activation of KRAS-MEK-ERK signaling. We hypothesize that this may be a mechanism for cells to sense the growth factor milieu and send that information to other cells via alterations in Ago2 and miRNA secretion.

McKenzie et al. “KRAS-MEK signaling controls Ago2 sorting into exosomes.” Cell Reports AOP 21 April 2016.