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).
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.
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.
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
Interactive Pathway Finder viewer on Genboree Workbench
This blog post is adapted from a TGen press release, found here.
Uncovering the genetic makeup of patients using DNA sequencing has in recent years provided physicians and their patients with a greater understanding of how best to diagnose and treat the diseases that plague humanity. This is the essence of precision medicine.
Now, researchers at the Translational Genomics Research Institute (TGen) are showing how an even more detailed genetic analysis using RNA sequencing can vastly enhance that understanding, providing doctors and their patients with more precise tools to target the underlying causes of disease and help recommend the best course of action.
In their review, published recently in the journal Nature Reviews Genetics, TGen scientists highlight the many advantages of using RNA-sequencing in the detection and management of everything from cancer to infectious diseases such as Ebola and the rapidly spreading Zika virus.
RNA’s principal role is to act as a messenger carrying instructions from DNA for the synthesis of proteins. Building on the insights provided by DNA profiling, the analysis of RNA provides an even more precise look at how cells behave and how medicine can intervene when things go wrong.
Dr. Sara Byron
“RNA is a dynamic and diverse biomolecule with an essential role in numerous biological processes,” said Dr. Sara Byron, Research Assistant Professor in TGen’s Center for Translational Innovation and the review’s lead author. “From a molecular diagnostic standpoint, RNA-based measurements have the potential for broad application across diverse areas of human health, including disease diagnosis, prognosis, and therapeutic selection.”
DNA (deoxyribonucleic acid) sequencing spells out — in order — the billions of chemical letters that make up the genes that drive all of our biologic make-up and functions, from hair and eye color to whether an individual may be predisposed to cancer or other diseases.
RNA (ribonucleic acid) sequencing provides information on the genes that are actively being made into RNA in a cell and are important for cell function. While more complex, RNA holds the promise of more precise measurement of the human physical condition.
There are more forms of RNA than of DNA present in the body, explains Dr. Byron. “RNA sequencing provides a deeper view of a patient’s genome, revealing detailed information on the diverse spectrum of RNAs being expressed.”
One of the most promising aspects of RNA-based measurements is the potential of using extracellular RNA (exRNAs) as a non-invasive diagnostic indicator of disease. Monitoring exRNA simply takes a blood sample, as opposed to doing a tumor biopsy, which is essentially a minor surgery with greater risks and costs.
“The investigation of exRNAs in biofluids to monitor disease is an area of diagnostic research that is growing rapidly,” said Dr. Kendall Van Keuren-Jensen, TGen Associate Professor of Neurogenomics, Co-Director of TGen’s Center for Noninvasive Diagnostics, and one of the review’s authors. “Measurement of exRNA is appealing as a non-invasive method for monitoring disease. With increased access to biofluids, more frequent sampling can occur over time.”
The first clinical test to measure exRNA was released earlier this year, the review said. The test is for use in evaluating lung cancer progression, and the potential for using RNA-seq in other cancers is expanding rapidly. Commercial RNA-seq tests are now available, providing the opportunity for clinicians to more comprehensively profile cancer and use this information to guide treatment selection for their patients, the review said.
In addition, the authors reported on several recent applications for RNA-seq in the diagnosis and management of infectious diseases, such as monitoring for drug-resistant populations during therapy and tracking the origin and spread of the Ebola virus.
Using examples from discovery and clinical research, the authors also describe how RNA-seq can guide interpretation of genomic DNA sequencing results. The use of integrative sequencing strategies in research studies is growing across a broad range of health applications, which promises to drive the incorporation of RNA-seq into clinical medicine as well, the review said.