RNA editing and immune control: from mechanism to therapy.

Adenosine-to-inosine RNA editing, catalyzed by the enzymes ADAR1 and ADAR2, stands as a pervasive RNA modification. A primary function of ADAR1-mediated RNA editing lies in labeling endogenous double-stranded RNAs (dsRNAs) as 'self', thereby averting their potential to activate innate immune responses. Recent findings have highlighted additional roles of ADAR1, independent of RNA editing, that are crucial for immune control. Here, we focus on recent progress in understanding ADAR1's RNA editing-dependent and -independent roles in immune control. We describe how ADAR1 regulates various dsRNA innate immune receptors through distinct mechanisms. Furthermore, we discuss the implications of ADAR1 and RNA editing in diseases, including autoimmune diseases and cancers.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

Voices: Challenges and opportunities in RNA biology.

In the first of many thematic issues marking the 30th anniversary of Cell Chemical Biology, we highlight the contribution of chemical biology to RNA biology in a special issue on RNA modulation. We asked several leaders in the field to share their opinions on the current challenges and opportunities in RNA biology.

ADAR1p150 prevents MDA5 and PKR activation via distinct mechanisms to avert fatal autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign nucleic acids from cellular ones. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA-editing enzyme ADAR1 to evade being recognized as viral dsRNA by cytoplasmic dsRNA sensors, including MDA5 and PKR. The loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. Additional RNA-editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, whereas loss of the cytoplasmic ADAR1p150 isoform or its dsRNA-binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5 or PKR alone. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

A-to-I RNA editing by ADAR and its therapeutic applications: From viral infections to cancer immunotherapy.

ADAR deaminases catalyze adenosine-to-inosine (A-to-I) editing on double-stranded RNA (dsRNA) substrates that regulate an umbrella of biological processes. One of the two catalytically active ADAR enzymes, ADAR1, plays a major role in innate immune responses by suppression of RNA sensing pathways which are orchestrated through the ADAR1-dsRNA-MDA5 axis. Unedited immunogenic dsRNA substrates are potent ligands for the cellular sensor MDA5. Upon activation, MDA5 leads to the induction of interferons and expression of hundreds of interferon-stimulated genes with potent antiviral activity. In this way, ADAR1 acts as a gatekeeper of the RNA sensing pathway by striking a fine balance between innate antiviral responses and prevention of autoimmunity. Reduced editing of immunogenic dsRNA by ADAR1 is strongly linked to the development of common autoimmune and inflammatory diseases. In viral infections, ADAR1 exhibits both antiviral and proviral effects. This is modulated by both editing-dependent and editing-independent functions, such as PKR antagonism. Several A-to-I RNA editing events have been identified in viruses, including in the insidious viral pathogen, SARS-CoV-2 which regulates viral fitness and infectivity, and could play a role in shaping viral evolution. Furthermore, ADAR1 is an attractive target for immuno-oncology therapy. Overexpression of ADAR1 and increased dsRNA editing have been observed in several human cancers. Silencing ADAR1, especially in cancers that are refractory to immune checkpoint inhibitors, is a promising therapeutic strategy for cancer immunotherapy in conjunction with epigenetic therapy. The mechanistic understanding of dsRNA editing by ADAR1 and dsRNA sensing by MDA5 and PKR holds great potential for therapeutic applications. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease.

ADAR1p150 Prevents MDA5 and PKR Activation via Distinct Mechanisms to Avert Fatal Autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign (non-self) nucleic acids from cellular (self) nucleic acids. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern being recognized as viral dsRNA by cytoplasmic dsRNA sensors including MDA5, PKR and ZBP1. A loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. However, additional RNA editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, while loss of the cytoplasmic ADAR1p150 isoform or its dsRNA binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150 -/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5, PKR or ZBP1 alone, demonstrating that this is a species conserved function of ADAR1p150. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2.

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory T cells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.

RNA editing restricts hyperactive ciliary kinases.

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis.

Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs. Despite a compelling need towards predictive understanding of natural and engineered editing events, how the RNA sequence and structure determine the editing efficiency and specificity (i.e., cis-regulation) is poorly understood. We apply a CRISPR/Cas9-mediated saturation mutagenesis approach to generate libraries of mutations near three natural editing substrates at their endogenous genomic loci. We use machine learning to integrate diverse RNA sequence and structure features to model editing levels measured by deep sequencing. We confirm known features and identify new features important for RNA editing. Training and testing XGBoost algorithm within the same substrate yield models that explain 68 to 86 percent of substrate-specific variation in editing levels. However, the models do not generalize across substrates, suggesting complex and context-dependent regulation patterns. Our integrative approach can be applied to larger scale experiments towards deciphering the RNA editing code.

Zinc Finger RNA-Binding Protein Zn72D Regulates ADAR-Mediated RNA Editing in Neurons.

Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. Here, we screen RNA-binding proteins for roles in editing regulation with knockdown experiments in the Drosophila brain. We identify zinc-finger protein at 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and fly mobility. Furthermore, Zn72D's regulatory role in RNA editing is conserved because the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons. The broad and conserved regulation of ADAR editing by Zn72D in neurons sustains critically important editing events.

Unbiased Identification of trans Regulators of ADAR and A-to-I RNA Editing.

Adenosine-to-inosine RNA editing is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes that deaminate adenosine to inosine. Although many RNA editing sites are known, few trans regulators have been identified. We perform BioID followed by mass spectrometry to identify trans regulators of ADAR1 and ADAR2 in HeLa and M17 neuroblastoma cells. We identify known and novel ADAR-interacting proteins. Using ENCODE data, we validate and characterize a subset of the novel interactors as global or site-specific RNA editing regulators. Our set of novel trans regulators includes all four members of the DZF-domain-containing family of proteins: ILF3, ILF2, STRBP, and ZFR. We show that these proteins interact with each ADAR and modulate RNA editing levels. We find ILF3 is a broadly influential negative regulator of editing. This work demonstrates the broad roles that RNA binding proteins play in regulating editing levels, and establishes DZF-domain-containing proteins as a group of highly influential RNA editing regulators.

Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila.

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.

Global landscape and genetic regulation of RNA editing in cortical samples from individuals with schizophrenia.

RNA editing critically regulates neurodevelopment and normal neuronal function. The global landscape of RNA editing was surveyed across 364 schizophrenia cases and 383 control postmortem brain samples from the CommonMind Consortium, comprising two regions: dorsolateral prefrontal cortex and anterior cingulate cortex. In schizophrenia, RNA editing sites in genes encoding AMPA-type glutamate receptors and postsynaptic density proteins were less edited, whereas those encoding translation initiation machinery were edited more. These sites replicate between brain regions, map to 3'-untranslated regions and intronic regions, share common sequence motifs and overlap with binding sites for RNA-binding proteins crucial for neurodevelopment. These findings cross-validate in hundreds of non-overlapping dorsolateral prefrontal cortex samples. Furthermore, ~30% of RNA editing sites associate with cis-regulatory variants (editing quantitative trait loci or edQTLs). Fine-mapping edQTLs with schizophrenia risk loci revealed co-localization of eleven edQTLs with six loci. The findings demonstrate widespread altered RNA editing in schizophrenia and its genetic regulation, and suggest a causal and mechanistic role of RNA editing in schizophrenia neuropathology.

ADAR1: A New Target for Immuno-oncology Therapy.

Three recent studies by Ishizuka et al. (2019), Liu et al. (2019), and Gannon et al. (2018) show that deleting RNA editing enzyme ADAR1 could induce higher cell lethality and render tumor cells more vulnerable to immunotherapy, pinpointing ADAR1 as a new immuno-oncology target.

Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.

Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development.

The THO Complex Coordinates Transcripts for Synapse Development and Dopamine Neuron Survival.

Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C. elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.

Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs.

Molecular tools that target RNA at specific sites allow recoding of RNA information and processing. SNAP-tagged deaminases guided by a chemically stabilized guide RNA can edit targeted adenosine to inosine in several endogenous transcripts simultaneously, with high efficiency (up to 90%), high potency, sufficient editing duration, and high precision. We used adenosine deaminases acting on RNA (ADARs) fused to SNAP-tag for the efficient and concurrent editing of two disease-relevant signaling transcripts, KRAS and STAT1. We also demonstrate improved performance compared with that of the recently described Cas13b-ADAR.