Unlocking the
Mysteries of
Extracellular RNA
Communication

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.

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.

Sample output from Target Interaction Finder

Sample output from Target Interaction Finder

We recently developed two tools for the Genboree Workbench: Target Interaction Finder and Pathway Finder. Given a set of miRNAs, these tools find miRNA-target interactions and pathway targets from public databases. To support consortium scientists in using these tools, we have created two training videos which describe the necessary input file, how to use the tool, and downstream network visualization in Cytoscape for Target Interaction Finder and Pathway Finder. The videos are available in the collection of ERC consortium videos at YouTube and in the Resources/Presentations section of this website.

The tools are open to anyone with a (free) Genboree account and can be used with any arbitrary input list of miRNA identifiers or with public datasets available in the exRNA Atlas. Target Interaction Finder generates a network of miRNA and protein target interactions, which is returned as a tabular summary and an XGMML formatted network file. The network file can be imported into network visualization and analysis tools like Cytoscape. Pathway Finder generates a table of pathways containing the miRNA and/or their protein targets based on information from WikiPathways. Embedded in the results window of Pathway Finder is an interactive pathway viewer.

Sample Pathway Finder results file

Sample Pathway Finder results file

exRNA_WP_blog_tools-PF-interactive

Interactive Pathway Finder viewer on Genboree Workbench