RNAs scaffold complex molecular machines that regulate their stability, translation, modification, and subcellular localization. These complexes are composed of RNA-binding proteins (RBPs) and their interacting RNA and protein components, and their activities have a major impact on the amount and duration of the production of each mRNA’s corresponding protein. They also affect the RNA’s ability to avoid innate immune recognition as a potentially dangerous or foreign nucleic acid, an event that initiates an inflammatory cascade that mediates antiviral immunity. In the case of noncoding RNAs, the scaffolding of ribonucleoprotein complexes enables a panoply of functions. Some of these are fundamental and well known to all biologists, such as the activities of the spliceosome and ribosome, both of which are megadalton complexes with RNA at their core. Others, such as microRNA (miRNA)-directed inhibition of protein translation and mRNA stability, have only recently gained widespread understanding and remain the topic of intense research. For long noncoding RNAs and other emerging classes of noncoding transcripts, their molecular functions and biological effects are still in a phase of avid discovery.

This volume of immunologic reviews highlights the great breadth of RNA function and regulation in the immune system. Topics reviewed span adaptive and innate immunity, including RNA regulation in some non-hematopoietic or “non-immune” cell types that nevertheless play critical roles in immune responses. The reviews detail the impacts of RNA regulation for normal physiology and pathologic processes, including the critical role of RNA recognition in the initiation and execution of antiviral immunity. In addition, they raise many questions that remain to be tackled in future studies. Expanding our fundamental understanding of RNA regulation of immunity will continue to drive technological developments with applications in diagnostics, therapeutics, and vaccines.

RNA is the medium through which information stored in the genome is animated to produce the wide array of cell programs and behaviors that constitute the complex tissue and organ systems and environmental responsiveness essential for the development and health of multicellular organisms like human beings. Far from being a passive messenger in this process, RNA plays myriad functional roles and subjects to intensive regulation (Figure 1). RNA regulation begins co-transcriptionally in the nucleus, with alternative splicing and alternative polyadenylation increasing the diversity of transcripts and protein products that can be encoded by a single gene. Subsequently, RNA sequence and/or structure-dependent binding of miRNAs and RNA-binding proteins (RBPs) adds a layer of post-transcriptional control to networks of gene expression. Additional layers of complexity are introduced by an array of RNA modifications, including capping, methylation, A-to-I editing, and others that modulate RBP binding and RNA stability and function.

 

Together, these layers of post-transcriptional regulation shape gene expression, and thereby control cell programming and function in all tissue and organ systems, including the immune system. For example, RNA regulation enforces the temporal link between engagement of immunological sensors and the production of inflammatory proteins by endowing the mRNAs that encode those proteins with short half-lives. Indeed, the signaling pathways that mediate immune cell activation biochemically modify and functionally regulate RBPs, and this process is essential to prevent hyperactivity of both innate and adaptive immunity. Several reviews in this issue address one or several of these layers of post-transcriptional regulation in a variety of cell types and immunological contexts.

1 LAYERS OF RNA REGULATION CONTROL GENE EXPRESSION IN THE IMMUNE SYSTEM

The sequence of a transcript affects its post-transcriptional regulation and function, ultimately driving cellular fate decisions. Taking a unique evolutionary view, Wolkers and colleagues highlight major intrinsic features of mRNA sequences that define RNA stability and translational efficiency.1 This includes sequence conservation, UTR length, GC content, and codon usage. In part, these sequence features act by dictating binding of other regulatory RNA (such as miRNAs). These interactions are capable of either enhancing or reducing protein production, affecting diverse cellular processes in T cells including proliferation, survival, differentiation, effector cell function, and memory formation. While the primary nucleotide sequence holds tremendous information content, added layers of regulation through the incorporation of modified nucleotides, A-to-I editing, alternative splicing, and alternative polyadenylation also impact these interactions. Throughout the review, the authors highlight the power of systematic and transcriptome-wide analysis to uncover fundamental principles of RNA regulation. Much remains to be discovered in this exciting research space, and the power of predicting sequence-based effects on immune function could have profound impacts on both understanding and engineering immune responses.

Blake and Lynch provide a detailed review of the control of mRNA maturation by alternative splicing and alternative polyadenylation in the immune system.2 In both processes, the intricate combinatorial binding of RBPs to target sequences on nascent RNAs can guide or restrict core machinery to alter mRNA transcripts. In alternative splicing, this changes both the inclusion and boundaries of exons and introns, altering the primary amino acid sequence of proteins. In alternative polyadenylation, 3’UTRs are shortened or lengthened, which can exclude or include regulatory sequences that affect transcript localization, stability, and translation. Both alternative splicing and alternative polyadenylation are widespread; it is estimated that ~95% of genes undergo alternative splicing and ~50% of genes undergo alternative polyadenylation. These alterations in mRNA affect immune cell function and immune responses. CD45 and TLR4 are classic examples of mRNAs whose alternative splicing limits prolonged activation of signaling and promotes a return to homeostasis in T cells and macrophages, respectively. Similarly, alternative polyadenylation of mRNAs encoding immunoglobulin heavy chains is a well-known determinant of antibody secretion. The authors also explore new frontiers in the field including the regulation of cell death by these processes in immune cells, with continued advances in transcriptomics opening alternative splicing and alternative polyadenylation as a new avenue for basic science discovery and therapeutic targeting among the panoply of genes that dictate immunologic function.

Monticelli and colleagues explore the roles of RBPs and RNA methylation in regulating the function of myeloid cells and their associated immune responses.3 The authors focus attention on the regulation of mRNA transcripts by the RBPs TTP and HuR at AU-rich elements, and on Regnase family members at stem-loop hairpins. These RBPs destabilize or, in some cases, directly cleave proinflammatory transcripts such as those encoding TNF-α and IL-6. Myeloid lineage-specific depletion of these critical RBPs leads to major perturbations in immune homeostasis and unchecked inflammation. Not surprisingly, these protein-RNA regulatory interactions are tightly regulated. Post-translational modifications of RBPs including phosphorylation and ubiquitination alter RNA-binding affinity, activity, and localization. The authors also highlight the importance of N6-methylation of adenosines (m6A) in sculpting RNA regulation through the activity of RBP writers, readers, and erasers of this methylation mark. They provide a useful summary of available databases and tools that catalog transcriptome-wide RNA modifications, and highlight the widespread and varied expression of all of these RBP classes in myeloid lineage cells. Great opportunities remain for discovery of RNA regulation in myeloid cell-driven inflammation.

Intercellular exchange of RNAs recently emerged as an exciting new means of cell-to-cell communication. Pua and colleagues review the foundational work that established this rapidly growing area of research, and present the latest developments in the roles of extracellular RNA communication in the immune system.4 Extracellular RNAs (exRNAs) exist in several distinct forms that allow them to resist degradation by the RNases that permeate blood and other body fluids. Extracellular vesicles (EVs) are the best-understood carrier of exRNA. Recent research has elucidated the cell biology of EV formation and secretion, and started to uncover the mechanisms by which RNAs are selected and packaged for export as EV cargo. ExRNAs include mRNAs and various noncoding RNA species, with miRNAs being distinguished as a major class that is enriched among the exRNA in EVs. Inflammation alters exRNA profiles in body fluids, reflecting changes in the frequency and abundance of source cells, their secretion of EVs, and their expression of potential exRNA cargoes. These changes present opportunities for biomarker discovery in immune-mediated diseases and suggest functions for exRNA communication in inflammatory processes. Indeed, accumulating evidence indicates that secreted miRNAs can regulate gene expression in recipient cells that uptake EVs. Lipid nanoparticles for RNA delivery to immune cells are the technological backbone of recently approved SARS-CoV2 RNA vaccines. Better understanding of exRNA communication and further development of technologies that mimic or harness this endogenous biological process may provide new avenues for therapeutic innovation.

The innate immune response to viral infection is tightly controlled through multiple layers of post-transcriptional regulation. Savan and colleagues comprehensively review RNA regulation of viral sensing by RIG-I-like receptors (RLRs) as well as interferon and interferon-stimulated genes that mediate antiviral immunity.5 Alternative splicing and polyadenylation, RNA modification, RBP-mediated regulation of mRNA translation and stability, miRNAs, and long noncoding RNAs are all engaged to regulate these pathways. The authors present an illuminating evolutionary perspective that highlights the arms race between host and viruses. RNA regulation is a common target of subversive mechanisms that determine viral pathogenesis. The authors also review specific examples of host natural variation that can determine the outcome of infection through altered RNA regulation of antiviral immunity. Clearly, the study of the interactions between viruses and their hosts is fertile ground for uncovering RNA-directed mechanisms and their impact on immunity.

The Regnase family of gene regulatory RBPs are remarkable for both their importance in maintaining immune homeostasis and their mechanism of action—direct endoribonucleolytic cleavage of target mRNAs. Mino and Takeuchi review current knowledge of this family with a focus on the founding member, Regnase-1.6 This protein plays a critical role in lymphocytes as well as epithelium and other non-immune cell types, where it recognizes stem-loop motifs in mRNAs that encode powerful inflammatory mediators. Regnase-1 itself is regulated in response to inflammatory signaling through post-translational modification and proteolytic cleavage and by the opposing activity of the RBP Arid5a. The authors also review the immunologic functions of the other 3 Regnase family members and discuss other endoribonucleases that function in antiviral immunity.

2 MICRORNAS REGULATE IMMUNITY

Koralov and colleagues explore the central role of miRNAs in B lymphocytes, with a special emphasis on how miRNAs regulate B cell receptor (BCR) signaling to affect development and function of these immune cells.7 Although VDJ recombination can still occur in globally miRNA-deficient developing B cells, changes in BCR repertoire diversity, BCR expression levels, non-templated nucleotide additions, and ongoing BCR editing in the periphery have been observed in these global miRNA knockout cells. Several signaling pathways are regulated by miRNAs in B cells, including PI3K-AKT, Ras-MAPK, and NF-kB pathways, and miRNAs post-transcriptionally inhibit genes with both positive and negative regulatory roles in these pathways. The authors highlight prominent roles for members of the miR-17~92 cluster, miR-148a, miR-29, and mIR-185 among others. By altering BCR signaling, miRNAs have significant impacts on B cell selection, tolerance, and cellular responses.

MicroRNAs control of CD4+ T helper cell differentiation and function has been a highly active area of research, with ever-expanding roles for miRNAs in both conventional and regulatory T cells. In their review, Lu and colleagues highlight recent studies examining how miRNAs impact T cells and their roles in the orchestration of immune responses and the maintenance of immune tolerance.8 The authors also explore the indirect effects that miRNA expression in other cell types can have on T cell biology. Not only can miRNAs impact classic T cell partners such as B cells, but they also control the behavior of cells that form tissue microenvironments that shape T cell responses, such as the intestinal epithelium and tumor cells.

Although noncoding RNAs in immune cells have broad impacts in shaping immune responses, Ansel and colleagues remind us that miRNA regulation of gene expression in non-hematopoietic cells can have broad impacts on tissue inflammation.9 They focus on the role of miRNAs in regulating the airway epithelium, and the critical role RNA-regulated gene expression plays in lung homeostasis and disease. Numerous studies have identified changes in miRNA expression in airway epithelium in diverse disease states, including asthma, chronic obstructive pulmonary disease (COPD), rhinitis, and cystic fibrosis. In epithelial cells, miRNA regulation of development, proliferation, differentiation, barrier integrity, and mucus production are intimately linked with the pathogenesis of inflammatory airway diseases. Among many examples, the authors discuss that the miR-34/449 family is reduced in the lungs of asthmatic patients, and that this miRNA family is required for normal mucociliary clearance in the lung. This miRNA family is also downregulated upon treatment of epithelial cells by the type 2 cytokine IL-13, suggesting possible linkages between inflammation, epithelial dysfunction, and miRNAs in allergic lung pathogenesis. In addition, miRNA expression in epithelial cells also helps directly regulate the cross-talk between lung epithelial and immune cells during tissue inflammation. Multiple miRNAs either directly or indirectly inhibit the production of epithelial cytokines including IL-33 and IL-8 that are critical for recruiting and activating local inflammatory responses. Importantly, the authors point out that not only are miRNAs capable of rewiring cellular responses that can alter the course of an inflammatory process, but that epithelial cells lining the airway are directly accessible for drug targeting through the inhalation route making them an attractive therapeutic target.

3 RNA RECOGNITION AND ANTIVIRAL IMMUNITY

Pyle and colleagues provide a detailed review of RNA-mediated RIG-I activation.10 RIG-I protein is a cytoplasmic pattern recognition receptor (PRR) that triggers early innate immune responses to viral infection. Within the cell, RIG-I sits in an inactive form, and transitory binding of host RNAs is insufficient to induce activation. However, RIG-I has special affinity for viral RNAs, especially 5’ di- and tri-phosphorylated blunt-ended RNA duplexes. Upon binding of these pathogen RNAs, RIG-I sounds a cellular alarm through the initiation of a potent signaling cascade to induce an interferon-mediated antiviral response. The determinants of this recognition depend on structure-function relationships between RIG-I and its RNA targets as well as protein-protein interactions. This relationship provides a uniquely mechanistic insight into innate immune responses and a model of the molecular consequences of RNA binding to an RBP. The authors highlight how this mechanism evolved to address the difficult task of identifying pathogen RNAs which exist within a sea of host RNA, and the danger of inappropriate activation of RIG-I if exquisite selectivity is not maintained, as exemplified by rare inborn errors of immunity. Finally, they discuss other PRRs that recognize RNA and help to diversify the cellular defense system against invading pathogens.

Horner and colleagues review antiviral immunity with a focus on the impacts of RNA modification on this process.11 The 5’ cap, long appreciated for its importance in supporting mRNA splicing, stability, nuclear export, and translation, also plays a key role in innate immune recognition. Viral mRNAs with aberrant or incomplete caps are bound by PRRs that mediate antiviral immunity. Not surprisingly, some viruses have evolved mechanisms to cap their RNAs to evade immune recognition. The authors also review the effects of the m6A modification, which also suppresses innate recognition by PRRs in addition to its roles in the regulation of host gene expression. Similarly, A-I editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes prevents host RNAs from activating innate immune responses. ADAR1 deficiency causes Aicardi-Goutiéres syndrome, a disease marked by constitutively active interferon responses. New and understudied RNA modifications with potential regulatory functions are also discussed. Clearly, RNA modification is a multifunctional and extremely important facet of RNA regulation in the immune system. As pointed out by Horner and colleagues, the discovery that incorporating modified bases into antigen-encoding mRNAs both increases protein production and limits innate immune recognition of foreign mRNA was essential to the success of the BTN162b2 and mRNA-1273 vaccines against SARS-CoV-2.

Amidst the chaos and tragedy of the past two years, RNA immunology emerged as a seemingly unlikely hero of the COVID-19 pandemic. Yet, it was several decades of fundamental and applied research that culminated in the astonishingly rapid development of mRNA vaccines for SARS-CoV-2, a novel pathogen that poses a worldwide threat. Our understanding of RNA biology and RNA-induced inflammatory responses paved the way to the deployment of vaccines with outstanding safety and efficacy in providing protection against severe COVID-19 disease and mortality. This triumph of immunology, RNA biology, and medicine will inspire scientists to continue to ask probing questions about RNA regulation in immunity, accelerating discovery and technology development that may drive creative solutions to other difficult challenges in science and medicine.

REFERENCES

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