Cells choose their words wisely.

In their recent Nature paper, Garcia-Martin et al. show that sequences within a microRNA influence how much of that microRNA is sent to another cell through extracellular vesicles. This supports a growing body of data demonstrating that cells use RNA to talk, but we know much less about how they listen.

The Nature and Nurture of Extracellular Vesicle-Mediated Signaling.

In the last decade, it has become clear that extracellular vesicles (EVs) are a ubiquitous component of living systems. These small membrane-enclosed particles can confer diverse functions to the cells that release, capture, or coexist with them in an environment. We use examples across living systems to produce a conceptual framework that classifies three modes by which EVs exert their functions: (a) EV release that serves a function for producing cells, (b) EV modification of the extracellular environment, and (c) EV interactions with, and alteration of, receiving cells. We provide an overview of the inherent properties of EVs (i.e., their nature) as well as factors in the environment and receiving cell (i.e., nurture) that determine whether transmission of EV cargo leads to functional cellular responses. This review broadens the context for ruminating on EV functions and highlights the emergent properties of EVs that define their role in biology and will shape their applications in medicine.

RNA-RNA interactions between respiratory syncytial virus and miR-26 and miR-27 are associated with regulation of cell cycle and antiviral immunity.

microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity.

miR-7 is recruited to the high molecular weight RNA-induced silencing complex in CD8+ T cells upon activation and suppresses IL-2 signaling.

Increasing evidence suggests mammalian Argonaute (Ago) proteins partition into distinct complexes within cells, but there is still little biochemical or functional understanding of the miRNAs differentially associated with these complexes. In naïve T cells, Ago2 is found almost exclusively in low molecular weight (LMW) complexes which are associated with miRNAs but not their target mRNAs. Upon T-cell activation, a proportion of these Ago2 complexes move into a newly formed high molecular weight (HMW) RNA-induced silencing complex (RISC), which is characterized by the presence of the GW182 protein that mediates translational repression. Here, we demonstrate distinct partitioning of miRNAs and isomiRs in LMW versus HMW RISCs upon antigen-mediated activation of CD8+ T cells. We identify miR-7 as highly enriched in HMW RISC and demonstrate that miR-7 inhibition leads to increased production of IL-2 and up-regulation of the IL-2 receptor, the transferrin receptor, CD71 and the amino acid transporter, CD98. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signaling and the metabolic processes regulated by IL-2.

Extracellular vesicles from malaria-infected red blood cells: not all are secreted equal.

Extracellular vesicles (EVs) mediate the transfer of molecules between cells and play diverse roles in host-pathogen interactions. Malaria is an important disease caused by intracellular Plasmodium species that invade red blood cells and these red blood cells release EVs. The EVs from infected cells have diverse functions in the disease and an obstacle in understanding how they exert their functions is that multiple EV types exist. In this issue of EMBO reports, Abou Karam and colleagues use sophisticated biophysical techniques to isolate and characterize two EV subpopulations produced by red blood cells infected with Plasmodium falciparum (Abou Karam et al, 2022). The authors show that these EV subpopulations have distinct sizes, protein content, membrane packing, and fusion capabilities, suggesting that EV subpopulations from infected cells could target different cell types and subcellular locations. This work underscores the concept that understanding EV heterogeneity will go hand in hand with understanding EV functions.

Disentangling sRNA-Seq data to study RNA communication between species.

Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host-pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.

Intracellular redox potential is correlated with miRNA expression in MCF7 cells under hypoxic conditions.

Hypoxia is a ubiquitous feature of cancers, encouraging glycolytic metabolism, proliferation, and resistance to therapy. Nonetheless, hypoxia is a poorly defined term with confounding features described in the literature. Redox biology provides an important link between the external cellular microenvironment and the cell's response to changing oxygen pressures. In this paper, we demonstrate a correlation between intracellular redox potential (measured using optical nanosensors) and the concentrations of microRNAs (miRNAs) involved in the cell's response to changes in oxygen pressure. The correlations were established using surprisal analysis (an approach derived from thermodynamics and information theory). We found that measured redox potential changes reflect changes in the free energy computed by surprisal analysis of miRNAs. Furthermore, surprisal analysis identified groups of miRNAs, functionally related to changes in proliferation and metastatic potential that played the most significant role in the cell's response to changing oxygen pressure.

Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides.

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here, we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.

Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells.

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster.

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

RNA-mediated communication between helminths and their hosts: The missing links.

Small RNAs have been discovered in a wide variety of extracellular environments and are now thought to participate in communication between cells and even between different organisms and species. Helminths are parasitic worms that generally reside in extracellular niches in their hosts and can establish chronic infection through the release of immunomodulatory factors. Recent work has demonstrated that Extracellular RNA (exRNA) may be another class of immunomodulator secreted by helminths. Here we will detail what is known about small RNA pathways in helminth pathogens (focusing on nematodes) and mammalian hosts. We will then explore the computational challenges with identifying RNA-RNA interactions between 2 different species and the paradigm of RNA-RNA co-evolution that accompanies this. Finally we explore the lingering questions that require further investigation to understand the properties of exRNA that would enable it to function as an immunomodulator.

RNA-mediated degradation of microRNAs: A widespread viral strategy?

Regulation of small RNAs by other non-coding RNAs is a ubiquitous feature of gene regulatory systems that can be exploited by viruses. Examples of this have been described in 3 different herpesviruses, where viral non-coding RNAs bind to highly abundant cellular (miRNAs), mediating their degradation: miR-27 is targeted by both murine cytomegalovirus and herpesvirus saimiri, while the miR-17 family is targeted by human cytomegalovirus. We review what is known about RNA-mediated regulation of miRNA stability and propose 3 potential roles that viral non-coding RNAs might assume to initiate the destruction of a miRNA, acting as "recruiters," "localizers" or "exposers." Whereas the miRNAs (miR-17 and miR-27) appear to be ancient and pre-date the common ancestor of all mammalian herpesviruses, comparative analyses of herpesvirus genomes indicate that the 3 known viral regulators of miRNA each evolved independently, and much more recently. Noting that the anti-viral activity of miRNAs might be countered by a variety of mechanisms, we propose that (i) there has been continual turnover of these mechanisms during herpesvirus evolution, and (ii) there may be many other, as yet undescribed, anti-miRNA activities encoded by other herpesviruses and indeed by viruses from other families.

Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target.

Individual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3'UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus-host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.

Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo.

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Regulation of microRNA biogenesis and turnover by animals and their viruses.

MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.

Intracellular localisation and extracellular release of Y RNA and Y RNA binding proteins.

Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners.

SID-2 is a conserved extracellular vesicle protein that is not associated with environmental RNAi in parasitic nematodes.

In the free-living nematode Caenorhabditis elegans, the transmembrane protein SID-2 imports double-stranded RNA into intestinal cells to trigger systemic RNA interference (RNAi), allowing organisms to sense and respond to environmental cues such as the presence of pathogens. This process, known as environmental RNAi, has not been observed in the most closely related parasites that are also within clade V. Previous sequence-based searches failed to identify sid-2 orthologues in available clade V parasite genomes. In this study, we identified sid-2 orthologues in these parasites using genome synteny and protein structure-based comparison, following identification of a SID-2 orthologue in extracellular vesicles from the murine intestinal parasitic nematode Heligmosomoides bakeri. Expression of GFP-tagged H. bakeri SID-2 in C. elegans showed similar localization to the intestinal apical membrane as seen for GFP-tagged C. elegans SID-2, and further showed mobility in intestinal cells in vesicle-like structures. We tested the capacity of H. bakeri SID-2 to functionally complement environmental RNAi in a C. elegans SID-2 null mutant and show that H. bakeri SID-2 does not rescue the phenotype in this context. Our work identifies SID-2 as a highly abundant EV protein whose ancestral function may be unrelated to environmental RNAi, and rather highlights an association with extracellular vesicles in nematodes.

Functional diversification of Argonautes in nematodes: an expanding universe.

In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.

Combined agonist-antagonist genome-wide functional screening identifies broadly active antiviral microRNAs.

Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.