Significance of exRNA Research
All cellular organisms secrete RNAs. The functions of these extracellular RNAs (exRNAs), however, are poorly understood. The two most likely functions of exRNAs are intercellular communication and interkingdom communication, as the characteristics of RNA make it a great agent for communication. RNA is information-dense, ephemeral, and is common to all forms of life. Knowing how to manipulate exRNA, and thus be able to influence the intracellular and interkingdom communications, will advance both agriculture and medicine through the development of new environmentally friendly pesticides, and new diagnostic and therapeutic tools for early detection and/or treatment of disease.
Although there is extensive literature on exRNAs in mammals (including a whole journal titled ExRNA!), we still know very little about how exRNAs are selected for secretion, how they are taken up by recipient cells, or their biological roles. We know even less about exRNAs produced by plants and insects, although exRNAs clearly have the potential to play a central role in inter-species communication. For example, a bacterial bee gut symbiont was recently engineered to express a double-stranded RNA (dsRNA) that targets parasitic mite genes. Remarkably, this RNA accumulated in the bee hemolymph, where no bacteria are present, and was taken up by the mites, inhibiting their growth; this potentially provides a new exRNA-based alternative to traditional miticides for protecting beehives.
Recent papers showed that microRNAs (miRNAs) and small interfering RNAs (siRNAs) secreted by both plants and mammals can be taken up by bacteria in their microbiomes and alter gene expression in a sequence-specific manner. Importantly for this proposal, mutations in host plants or mammals that inhibit the production of these RNAs cause major changes in their microbiomes. In mice, these changes can be rescued by feeding mice RNA purified from wild-type mouse feces, which indicates that gut microbes take up naked RNA.
Here, we propose that exRNA-based communication goes beyond miRNAs and siRNAs. Analyses of exRNAs in plants and animals have established that miRNAs and siRNAs account for only a tiny fraction of total exRNA, with many long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and tRNA fragments (tRFs) being far more abundant suggesting that exRNA-based communication involves more than just small RNAs.
In plants, most of what we know about exRNAs has come from work done by PIs Innes and Baldrich. That work has shown that extracellular wash fluids isolated from Arabidopsis leaves contain RNAs ranging from ~12 to 500 nucleotides (nts) (Fig. 1A) and include miRNAs, siRNAs, tRFs, rRNA fragments, tRNAs, and circRNAs. A large fraction of the RNAs longer than 50 nt appear to be circular in structure, based on their resistance to digestion by RNase R and on the presence of junction fragments indicative of circle formation. Notably, few full-length mRNAs were observed, suggesting that most plant exRNAs do not code for proteins. Also of note, nearly all of this RNA was found outside of extracellular vesicles (EVs) but protected against degradation by association with RNA-binding proteins. This finding is consistent with work in mammalian systems, which has shown that most exRNAs are located outside of vesicles [10]. This is an important observation, as it suggests that intercellular transport and delivery of RNAs may not require EVs.
Given our finding that plant cells secrete RNA and numerous reports that show mammalian cells secrete RNA, we asked whether insects also secrete RNA and whether it was qualitatively similar to other taxa. To rapidly answer this question, we collaborated with the Drosophila Genomics Resource Center at Indiana University, which provided us with Drosophila ovary epithelial cell cultures. We isolated total RNA from conditioned media, from cells, and from media that lacked cells and found that conditioned media contained abundant RNA species similar in overall sizes to our plant exRNAs (~18 to 300 nt) (Fig. 1B). This pattern was completely different from that of total cell lysate or cell-free medium, indicating that this RNA is a product of secretion rather than cell death.
Further confirmation that these exRNAs are truly secreted comes from our analyses of N6methyladenosine (m6A) abundance, which is a post-transcriptional modification that has been linked to the secretion of miRNAs and circRNAs by mammalian cells (e.g., m6A modification of circNSUN2 increases its cytoplasmic export). Using dot blots and an anti-m6A antibody, we found that exRNA-isolated from Arabidopsis leaves is highly enriched in m6A compared to RNA isolated from whole cell lysates (Fig. 1C), further suggesting that post-transcriptional modifications may tag RNAs for export.