RNA regulation of the immune system

This article introduces a series of reviews covering RNA Regulation of the Immune System appearing in volume 253 of Immunological Reiews. RNA is involved in the expression of all genes. Messenger RNAs (mRNAs) are transcribed from the DNA genome and translated into protein by the ribosome. Not surprisingly, every step in this process is regulated to tune gene expression to meet the demands of complex organism development and appropriate responses to cell stress and environmental stimuli. Noncoding RNAs are major participants in gene regulation. The ribosome itself an RNA enzyme, and mRNA splicing is also mediated by small nuclear RNAs and their associated proteins. MicroRNAs (miRNAs) have recently emerged as essential and nearly ubiquitous regulators of gene expression in multicellular eukaryotes. Advanced sequencing technologies and the excitement surrounding the discovery of miRNAs and their myriad roles in a wide variety of biological systems (not least the immune system) has also reinvigorated research on other aspects of RNA biology. It is now clear that even transcription is subject to regulation by long noncoding RNAs. With the realization that over half of the genome is transcribed, we can expect additional revelations about RNA regulation in years to come. This volume of Immunological Reviews focuses on RNA regulation of the immune system, with a heavy dose of miRNAs and a sampling of other key processes, including alternative splicing, RNA-binding proteins that control mRNA stability and translation, and attempts to harness the miRNA machinery for therapeutic ends through RNA interference (RNAi). Conspicuously and intentionally underrepresented due to space constraints are two topics that deserve issues of their own: mechanisms of transcriptional regulation and immune recognition of RNA and its role in genome defense, host defense, and autoimmunity. The authors have all made important contributions to our understanding of RNA regulation of the immune system and in some cases have pioneered and defined whole sectors of the field. Major topics of their reviews are indicated using the initials of each corresponding author in Fig. 1. The figure also provides a simplified schematic overview of RNA regulation of gene expression within a cell and its effects on cell biology and emergent properties of the immune system. miRNAs first emerged from forward genetic screens for genes involved in temporal control of developmental events in Caenorhabtidis elegans. The first miRNA to be discovered, lin-4, controls worm development and acts as a repressor of the protein-coding gene lin-14 1. As evidence accumulated that lin-4 encodes a short RNA that regulates multiple genes, the discovery of RNAi in plants and worms hinted at the existence of deeply conserved mechanisms of gene regulation mediated by endogenous short RNAs 2, 3. Soon, a second C. elegans miRNA with a similar function in developmental timing was discovered 4. The realization that the human genome (and many others) encoded let-7 homologues 5 set off a sustained frenzy of discoveries that linked the mechanisms and machinery of RNAi and miRNA regulation of gene expression 6, elucidated the biogenesis pathway that processes miRNAs into their mature form 7, and uncovered the existence of hundreds of unique miRNAs – well over 1000 in humans at the last count 8. miRNAs' myriad roles in biology have attracted investigators from across the research spectrum, propelling work from the most basic investigation to clinical application in diagnostics and therapeutics 9. The miRNA pathway has essential functions in almost every cell and organ system in which it has been tested. Developmental transitions and cell differentiation remain a common theme, but miRNAs also critically regulate signaling and basic cellular functions such as proliferation and survival. miRNAs can be oncogenes or tumor suppressors, and their dysregulation strongly influences oncogenesis and metastasis. It has been noted that miRNA functions are often revealed only in the context of physiological or pathophysiological stresses associated with disease 10. This fits with the emerging concept that miRNAs confer robustness to biological process by tuning gene expression networks and repressing aberrant transcription events 11. miRNAs are short, endogenously expressed RNA molecules of approximately 21 nucleotides in length. They do not encode peptides, but instead regulate the expression of proteins encoded by their mRNA targets. With few exceptions, miRNAs share a common biogenesis pathway 7. Their primary transcripts ('pri-miRNAs') are transcribed from the genome in the ordinary way, usually by RNA polymerase II (Pol II). Some miRNAs share a transcript with a protein-coding gene, appearing in an intron or exonic 3′ untranslated region (UTR) of an mRNA, while other pri-miRNAs have dedicated promoters and contain no functional open reading frames. miRNAs often appear in 'clusters' in the genome. Although appearance in a cluster does not guarantee co-regulation of their expression, these miRNAs are often transcribed together in a single pri-miRNA. Like mRNAs, pri-miRNAs may be spliced or left unspliced, and those transcribed by Pol II are polyadenylated at their 3′ end. After this point, however, mRNAs and pri-miRNAs diverge in their path to maturity and function in the cytoplasm. Stem-loop structures containing the mature miRNA sequence are cropped out of pri-miRNAs by the Microprocessor complex, which consists of the RNAse III enzyme Drosha and its obligate RNA-binding protein partner Dgcr8. This yields 'pre-miRNAs' that are exported to the cytoplasm, where they are further processed by the related enzyme Dicer. Dicer removes the loop from pre-miRNAs, yielding a short double-stranded RNA very similar to the exogenous short-interfering RNAs (siRNAs) that mediate RNAi when transfected into mammalian cells. Indeed, one strand of this final miRNA precursor is loaded onto an Argonaute (Ago) protein, the core functional component of the RNA-induced silencing complex (RISC). Association with Ago stabilizes the miRNA, whereas the 'passenger' strand is short lived. miRNAs derived from the 5′ end of the stem-loop pre-miRNA are termed -5p (e.g. miR-142-5p), and those derived from the 3′ end are termed -3p (e.g. miR-142-3p). Thermodynamic features of the RNA duplex strongly influence strand selection, and most miRNA precursors display strong strand bias that results in only one functionally relevant miRNA. However, both strands of some precursors are loaded into Ago at similar rates, and strand selection can be modulated in a context-dependent fashion in cells of the immune system 12. Ago proteins programmed with a miRNA form the 'miRISC', and this complex is responsible for miRNA-directed gene repression 13. Ago proteins interact with factors that repress translation and accelerate mRNA decay, while the miRNA component provides sequence-specific target mRNA recognition through complementary base pairing 14. Targeted sequences may reside in any part of the mRNA, but bioinformatic, biochemical, and mechanistic studies indicate that target sequences in the 3′ UTR are the most common and most effective sites of miRISC action. Within the miRISC structure, nucleotides in positions 2–8 from the 5′ end of the miRNA are particularly accessible for base pairing 15, and this 'seed' sequence is a major determinant of targeting. Therefore, miRNA are classified into 'families' that share a common seed sequence and presumably a large fraction of their mRNA targets. However, targeting does not require perfect complementarity with the seed sequence, and further pairing in the 3′ half of the miRNA can enhance or even be sufficient alone to direct repression of some mRNAs. The complex degeneracy of miRNA target sequences and the redundancy provided by large seed families complicate target prediction and validation. Several target prediction algorithms have been devised and in some cases continuously refined using empirical data 16. These programs are very useful as a starting point for hypothesis generation in functional studies. However, they are generally limited either by their dependence on seed pairing and evolutionary conservation of miRNA and target sequences or by the large number of false positive predictions that result when these constraints are removed. Validation of these predictions is further complicated by the relatively modest repression mediated by miRNAs on each of their direct targets – almost always less than twofold at both the mRNA and protein level 17. Sensitive luciferase-based reporter assays are the current gold standard to test candidate miRNA targets, but these experiments often fail to preserve the cellular and sequence context in which the biologically relevant interaction is proposed to take place. Biochemical methods that detect Ago protein association with mRNAs have been developed and employed in the immune system 18-21. Assays like these will likely become part of the standard for evidence of direct miRNA targeting, as chromatin immunoprecipitation has become necessary to validate direct gene activation or repression by transcription factors. So how do such small regulators with such modest activity on each target have meaningful biological effects? This question underlies the important challenge of integrating miRNAs into our models of gene expression networks that govern cell identity and behavior. In some cases, a single target has been identified that can account for a significant proportion of the functional effect of a miRNA. 'Fine tuning' of gene expression matters, and small changes in the abundance of a limiting factor can trigger threshold effects and feedback loops that drive big changes in cell behavior. However, it is a common misconception that a key dominant target must exist. Like transcription factors, miRNAs function through their combined action on a large number of targets. Evolutionarily conserved miRNA target sequences occur in over half of all mRNAs, and each miRNA interacts with dozens to hundreds of mRNAs 22. The conservation of miRNA binding sequences in the untranslated regions of so many target mRNAs indicates that the modest effects mediated by many of these interactions are important enough to exert selection pressure in some evolutionarily relevant context. miRNAs often amplify their effects by targeting several genes that participate in a common pathway. This principle can be exploited for pathway discovery when the biological function and bona fide targets of a miRNA are known 23. Regulation of hematopoiesis in the bone marrow was the first established function of miRNAs in the immune system 24. Indeed, this was among the first demonstrations of any miRNA function in vertebrate physiology. The first miRNA expression profiling in purified primary cell populations was also conducted on hematopoietic precursors and their mature progeny 25. The miRNA pathway is essential for definitive hematopoiesis, and several individual miRNAs now have established roles in the development and function of the diverse immune cells that make up the adaptive and innate immune system. Monticelli et al. 26 review the role of miRNAs in mast cells, dendritic cells, and macrophages. These sentinel cells bridge innate and adaptive immunity, and the role of miRNAs in their development and immune function has been studied intensively. They are discussed here in the context of normal physiology and pathological states including mastocytosis, allergy, autoimmunity, and cancer. Ramiro et al. 27 review miRNA regulation of B-cell biology, including important early studies of early B-cell differentiation in the bone marrow and more recent research on miRNA control of antibody responses. miRNAs also regulate the maturation, homeostasis, and immune function of natural killer (NK) cells. Like many aspects of NK biology, this area has been pioneered by Lanier et al. Their review brings us up-to-date and draws insightful comparisons between miRNA regulation of NK cells and cytotoxic T cells 28. T cells have become an important experimental model system for the study of basic molecular mechanisms of gene regulation and cell differentiation. The depth of our understanding of the gene expression networks that govern T-cell lineage decisions and immune function facilitate integration of new players, such as miRNAs. Three reviews in this issue cover overlapping aspects of this extensive research area. The first, by Liston et al. 29, focuses first on thymocyte development, which they have shown requires miRNA activity not only in early stage developing thymocytes but also in the thymic epithelial cells that support their maturation and selection. They bring a similar perspective to the discussion of mature T cells, which are also subject to miRNA regulation in both the responding T cells themselves as well as in antigen-presenting cells and other accessory cells that modulate the activation and microenvironmental signals that shape their responses. Bluestone and Jeker 30 review current knowledge of miRNA regulation of cytotoxic T cells, helper T cells, and regulatory T cells. In so doing, they highlight several emerging principles of miRNA regulation and identify important challenges for further progress. Pagani et al. 31 put forward a network view of coding and non-coding RNA control of T-cell function. They emphasize the utility of high throughput sequencing technologies to determine RNA expression and interactions to elucidate these networks, and also introduce the budding area of long non-coding RNA regulation of gene expression. In many organisms, RNAi is a core component of host defense against invaders of the genome, such as viruses and transposons. Rana and Zhou 32 review RNA-based immunity in Drosophila and relate it to the evolutionary successors that mediate immune RNA sensing and genome defense in vertebrates. In addition, they discuss mechanisms by which viruses manipulate small regulatory RNA pathways to their own advantage. DNA viruses that infect vertebrates often manipulate host gene expression using virally encoded miRNAs, and the activity of host miRNAs can also be exploited by viruses. In contrast, viruses that infect flies and plants often encode inhibitors of RNAi to suppress RNA-based immune defenses. The absence of these mechanisms in viruses that infect vertebrates underscores the evolutionary shift from RNA-based immunity to other modes of RNA sensing and adaptive immunity. Nevertheless, the miRNA pathway continues to play myriad important roles in the development and function of the mammalian immune system. Wherever the requirement for miRNAs has been tested in the immune system, essential roles have been found. miRNAs regulate adaptive and innate immunity, autoimmunity and inflammation, and lymphomagenesis and leukemogenesis. Chen et al. 33 summarize recent advances in several of these areas, highlighting the importance of spatiotemporal regulation of immune signaling strength and thresholds by miRNAs. This mode of miRNA regulation is exemplified by their studies of the miR-181 family in thymocyte development, as well as by the interplay between miR-146a and the nuclear factor-κB (NF-κB) signaling pathway. NF-κB directly activates pri-miR-146a transcription, and mature miR-146a targets tumor necrosis factor receptor-associated factor 6 (TRAF6) and interferon receptor-associated kinase 1 (IRAK1), thereby attenuating further NF-κB signaling 34. Baltimore et al. 35 review and compare the functions of miR-146a and the mammalian lin-4 homologue miR-125b in normal immune function, inflammation, myeloproliferation, and cancer. They also discuss the relationship between miR-146a and miR-155, another miRNA that is potently induced by NF-κB. Unlike miR-146a, miR-155 amplifies signaling and is required for robust innate and adaptive immune responses. Vigorito et al. 36 discuss the evolutionary origins of miR-155, its function in lymphocytes and cancers of B-cell origin, and its potential as a target for therapy. Like miR-155, the miR-17∼92 cluster of miRNAs is often overexpressed in cancers, particularly (but not exclusively) those derived from immune cells. He et al. discovered the oncogenic potential of the polycistronic miR-17∼92 cluster 37, and their review 38 provides in-depth discussion of the complex regulation of its six miRNAs and the dissection of their many functions in normal physiology and tumorigenesis. Given their role as regulators of cell proliferation, survival, differentiation, and response to the environment, it should come as no surprise that miRNAs play an outsized role in cancer. Many of the reviews in this issue touch on the tumor suppressor and oncogenic effects of different miRNAs in various immune cells. Croce et al. have contributed over 250 published studies linking miRNAs and cancer biology. Here, they review 39 the oncogenic functions of five miRNAs that are also critical for responses to immune stimuli or hypoxia, linking inflammation and cancer. Again the possibility of developing miRNA-directed therapies for immune malignancies is discussed. As pointed out by Peer in his review 40, targeting RNA-based therapies to leukocytes is 'a daunting task'. Strategies for delivering small-interfering RNAs that harness the miRNA machinery to potently target specific mRNAs are considered, as is the possibility of delivering mimics of miRNAs to take advantage of their co-evolution with suites of targets in common biological pathways. Perhaps the simplest challenge is to antagonize the function of endogenous miRNAs, as this can be accomplished using single-stranded nucleic acid oligomers modified to increase stability and facilitate cell entry. Clinical development has advanced furthest in hepatic diseases, as systemic delivery concentrates these compounds in the liver 41. The lung is another viable target, as there are very well-established mechanisms for local delivery. Advances in our understanding of the role of the epithelial barrier and other accessory cells in pulmonary immunity expand the set of miRNAs that may be therapeutically relevant. Foster et al. have pioneered miRNA modulation in preclinical models of pulmonary disease, and their review 42 covers the role of miRNAs in host defense and immune dysfunction in the lung. Both pri-miRNAs and nascent mRNA transcripts require post-transcriptional processing before they can mediate their ultimate functions in gene expression and cell behavior. Each processing step is subject to regulation, as is the stability of mature miRNAs and mRNAs. Splicing and polyadenylation precede mRNA transport to the cytoplasm and translation into protein. Lynch has led pioneering studies of the cis- and trans-acting factors that control alternative splicing in the immune system and the role of this process in lymphocyte function. She and Martinez 43 review these topics and foresee a better understanding of the regulatory networks that coordinate alternative splicing of many genes to achieve particular biological effects in response to immune stimuli and a path to this understanding through the use of high throughput sequencing technology. Lieberman et al. 44 have found that there is a global disruption of RNA splicing and export that occurs during immune-mediated cell death. She and Thomas 45 review the many ways that RNA metabolism is regulated by the integrated stress response and discuss how these mechanisms influence the decision between repair and cell death. This includes the role of miRNAs in cell stress and the well-established role of RNA-binding proteins (RBPs) that regulate translation and mRNA stability. A diverse array of RBPs mediates powerful effects on gene expression in the immune system. Anderson and Ivanov 46 comprehensively review these RBPs, their mechanisms of action, crosstalk between these pathways and miRNAs, and their prominent role in autoimmunity. For example, sanroque mice that express a point mutant form of the RBP Roquin-1 develop spontaneous lupus-like autoimmune disease with a marked accumulation of follicular helper T cells 47. Heissmeyer and Vogel 48 focus on Roquin-1 and its recently renamed homologue Roquin-2 (formerly MNAB). They review recent advances in deciphering the molecular mechanism by which Roquin proteins maintain immune tolerance and propose a parsimonious model for the molecular and immunologic effects of the sanroque mutation. RNA-binding proteins are integral parts of miRNA biogenesis and function as well. In several cases, RBPs that regulate miRNA processing have also been found to act directly on other non-coding RNAs and/or mRNAs. Muljo and Yuan 49 discuss the pleiotropic functions of RBPs with a focus on Lin28. Lin28 proteins bind and inhibit the processing of let-7 precursors, but they also directly regulate some mRNAs. In the immune system, Lin28B is essential to suppress a fetal program in adult hematopoietic cells, consistent with a deeply conserved role in developmental transitions in animals 50. miRNA homeostasis is regulated at multiple levels in response to developmental and environmental cues. In the final review of this issue, Ansel and Bronevetsky 51 discuss miRNA and transcription factor feedback loops and RBP regulators of miRNA processing, including several well-known transcription factors that also bind and regulate pri-miRNAs. Finally, recent advances in our understanding of miRNA turnover and dynamic changes that occur during lymphocyte activation and cell stress are discussed. It seems that the immune system has taken every opportunity to regulate its gene products starting from the moment they are transcribed into RNA. Interactions between RNAs and a diverse array of RBPs form complex networks with powerful effects on gene expression. The discovery of miRNAs has ushered in a renaissance in the study of RNA regulation, and immunologists have uncovered a wide array of RNA-directed mechanisms that serve essential roles in immune system development and function. Some of these molecular mechanisms are viable targets for therapy in immune dysfunction and cancer. In any case, advances in our basic understanding of RNA regulation of the immune system can be expected to continue apace in coming years. This work was supported by the National Institutes of Health (P01HL107202, R01HL109102), and a Scholar Award from The Leukemia & Lymphoma Society (1593-13). The author has no conflicts of interest to declare.