Chilling with cephalopods: Temperature-responsive RNA editing in octopus and squid.

The molecular mechanisms that generate the developmental and physiological complexity found within cephalopods are not well understood. In this issue of Cell, Birk et al. and Rangan and Reck-Peterson show that cephalopods differentially edit their RNA in response to temperature changes and that this editing has consequences on protein function.

Temperature-dependent RNA editing in octopus extensively recodes the neural proteome.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

RGEN Editing of RNA and DNA: The Long and Winding Road from Catalytic RNAs to CRISPR to the Clinic.

The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.

Sort Your Self Out!

Discrimination between viral and self-derived nucleic acid species is crucial in maintaining effective antiviral immunity whilst avoiding autoinflammation. Ahmad et al. and Chung et al. delineate the consequences of MDA5 gain of function and loss of ADAR1 activity, highlighting the blurring of the concept of self and non-self when considering endogenous retroelements.

Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods.

RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals for this purpose. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of the nature and effects of these events. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system, affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein-coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function. PAPERCLIP.

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (targets of RNA-binding proteins identified by editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.

ADAR1 edits the SenZ and SenZ-ability of RNA.

Some RNAs can assume a Z conformation, an unusual, left-handed turn. In this issue of Immunity, three studies report that mutations in the Zα-RNA binding domain of the adenosine deaminase ADAR1 are sufficient to induce autoinflammatory disease in mice, which models human Aicardí-Goutières syndrome, highlighting the important role of Z-RNA editing in limiting innate immune recognition of endogenous RNA.

Mutations in the adenosine deaminase ADAR1 that prevent endogenous Z-RNA binding induce Aicardi-Goutières-syndrome-like encephalopathy.

Mutations in the adenosine-to-inosine RNA-editing enzyme ADAR1 p150, including point mutations in the Z-RNA recognition domain Zα, are associated with Aicardi-Goutières syndrome (AGS). Here, we examined the in vivo relevance of ADAR1 binding of Z-RNA. Mutation of W197 in Zα, which abolished Z-RNA binding, reduced RNA editing. Adar1W197A/W197A mice displayed severe growth retardation after birth, broad expression of interferon-stimulated genes (ISGs), and abnormal development of multiple organs. Notably, malformation of the brain was accompanied by white matter vacuolation and gliosis, reminiscent of AGS-associated encephalopathy. Concurrent deletion of the double-stranded RNA sensor MDA5 ameliorated these abnormalities. ADAR1 (W197A) expression increased in a feedback manner downstream of type I interferons, resulting in increased RNA editing at a subset of, but not all, ADAR1 target sites. This increased expression did not ameliorate inflammation in Adar1W197A/W197A mice. Thus, editing of select endogenous RNAs by ADAR1 is essential for preventing inappropriate MDA5-mediated inflammation, with relevance to the pathogenesis of AGS.

Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses.

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.

Targeted RNA editing: novel tools to study post-transcriptional regulation.

RNA binding proteins (RBPs) regulate nearly all post-transcriptional processes within cells. To fully understand RBP function, it is essential to identify their in vivo targets. Standard techniques for profiling RBP targets, such as crosslinking immunoprecipitation (CLIP) and its variants, are limited or suboptimal in some situations, e.g. when compatible antibodies are not available and when dealing with small cell populations such as neuronal subtypes and primary stem cells. This review summarizes and compares several genetic approaches recently designed to identify RBP targets in such circumstances. TRIBE (targets of RNA binding proteins identified by editing), RNA tagging, and STAMP (surveying targets by APOBEC-mediated profiling) are new genetic tools useful for the study of post-transcriptional regulation and RBP identification. We describe the underlying RNA base editing technology, recent applications, and therapeutic implications.

Deciphering the principles of the RNA editing code via large-scale systematic probing.

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.

Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination.

The AID/APOBEC polynucleotide cytidine deaminases have historically been classified as either DNA mutators or RNA editors based on their first identified nucleic acid substrate preference. DNA mutators can generate functional diversity at antibody genes but also cause genomic instability in cancer. RNA editors can generate informational diversity in the transcriptome of innate immune cells, and of cancer cells. Members of both classes can act as antiviral restriction factors. Recent structural work has illuminated differences and similarities between AID/APOBEC enzymes that can catalyse DNA mutation, RNA editing or both, suggesting that the strict functional classification of members of this family should be reconsidered. As many of these enzymes have been employed for targeted genome (or transcriptome) editing, a more holistic understanding will help improve the design of therapeutically relevant programmable base editors.

Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation.

The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.

A-to-I editing of coding and non-coding RNAs by ADARs.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.

Programmable RNA sensing for cell monitoring and manipulation.

RNA is a central and universal mediator of genetic information underlying the diversity of cell types and cell states, which together shape tissue organization and organismal function across species and lifespans. Despite numerous advances in RNA sequencing technologies and the massive accumulation of transcriptome datasets across the life sciences1,2, the dearth of technologies that use RNAs to observe and manipulate cell types remains a bottleneck in biology and medicine. Here we describe CellREADR (Cell access through RNA sensing by Endogenous ADAR), a programmable RNA-sensing technology that leverages RNA editing mediated by ADAR to couple the detection of cell-defining RNAs with the translation of effector proteins. Viral delivery of CellREADR conferred specific cell-type access in mouse and rat brains and in ex vivo human brain tissues. Furthermore, CellREADR enabled the recording and control of specific types of neurons in behaving mice. CellREADR thus highlights the potential for RNA-based monitoring and editing of animal cells in ways that are specific, versatile, simple and generalizable across organ systems and species, with wide applications in biology, biotechnology and programmable RNA medicine.

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.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

Genetics of cell-type-specific post-transcriptional gene regulation during human neurogenesis.

The function of some genetic variants associated with brain-relevant traits has been explained through colocalization with expression quantitative trait loci (eQTL) conducted in bulk postmortem adult brain tissue. However, many brain-trait associated loci have unknown cellular or molecular function. These genetic variants may exert context-specific function on different molecular phenotypes including post-transcriptional changes. Here, we identified genetic regulation of RNA editing and alternative polyadenylation (APA) within a cell-type-specific population of human neural progenitors and neurons. More RNA editing and isoforms utilizing longer polyadenylation sequences were observed in neurons, likely due to higher expression of genes encoding the proteins mediating these post-transcriptional events. We also detected hundreds of cell-type-specific editing quantitative trait loci (edQTLs) and alternative polyadenylation QTLs (apaQTLs). We found colocalizations of a neuron edQTL in CCDC88A with educational attainment and a progenitor apaQTL in EP300 with schizophrenia, suggesting that genetically mediated post-transcriptional regulation during brain development leads to differences in brain function.