APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

ADAR1-mediated RNA editing promotes B cell lymphomagenesis.

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancies. Here, we explore the contribution of RNA editing to DLBCL pathogenesis. We observed that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumors can modulate pathway outcomes by altering sequences at either the genomic or the transcriptomic level. RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as the RIG-I-like pathway. In this context, we show that ADAR1-mediated editing within MAVS transcript positively correlates with MAVS protein expression levels, associating with increased interferon/NF-κB signaling and T cell exhaustion. Finally, using targeted RNA base editing tools to restore editing within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing.

Harnessing self-labeling enzymes for selective and concurrent A-to-I and C-to-U RNA base editing.

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.

C-to-U RNA Editing: From Computational Detection to Experimental Validation.

The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.

A New Chapter in Genetic Medicine: RNA Editing and its Role in Disease Pathogenesis.

The transfer of genomic information from DNA to mRNA to protein usually occurs with high fidelity, but can also be subverted by a programmed RNA sequence alteration termed 'RNA editing', involving deamination of adenosine to inosine (decoded as guanosine), or of cytosine to uracil. These sequence changes can lead to cellular heterogeneity by generating variable sets of transcripts within otherwise identical cells. Recent studies have demonstrated that editing is most prevalent in cells and tissues with high propensity for plasticity. Within those, RNA editing reproducibly targets transcripts of related function, altering the outcomes of entire pathways at once. In ongoing work, changes in patterns of editing have been correlated with neuronal disease pathogenesis, suggesting that RNA editing harbors diagnostic potential.

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells.

Loss of APOBEC1 RNA-editing function in microglia exacerbates age-related CNS pathophysiology.

Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.

RNA editing generates cellular subsets with diverse sequence within populations.

RNA editing is a mutational mechanism that specifically alters the nucleotide content in transcribed RNA. However, editing rates vary widely, and could result from equivalent editing amongst individual cells, or represent an average of variable editing within a population. Here we present a hierarchical Bayesian model that quantifies the variance of editing rates at specific sites using RNA-seq data from both single cells, and a cognate bulk sample to distinguish between these two possibilities. The model predicts high variance for specific edited sites in murine macrophages and dendritic cells, findings that we validated experimentally by using targeted amplification of specific editable transcripts from single cells. The model also predicts changes in variance in editing rates for specific sites in dendritic cells during the course of LPS stimulation. Our data demonstrate substantial variance in editing signatures amongst single cells, supporting the notion that RNA editing generates diversity within cellular populations.

RNA Editing Dynamically Rewrites the Cancer Code.

Global analyses of cancer transcriptomes demonstrate that ADAR (adenosine deaminase, RNA-specific)-mediated RNA editing dynamically contributes to genetic alterations in cancer, and directly correlates with progression and prognosis. RNA editing is abundant and frequently elevated in cancer, and affects functionally and clinically relevant sites in both coding and non-coding regions of the transcriptome. Therefore, ADAR and differentially edited transcripts may be promising biomarkers or targets for therapy.

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding.

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.

A single zinc ion is sufficient for an active Trypanosoma brucei tRNA editing deaminase.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.

MicroRNA control of lymphocyte differentiation and function.

MicroRNAs (miRNAs) are a class of endogenous, non-coding regulatory RNAs that control gene regulation by guiding silencing protein complexes to mRNA in a sequence-dependent manner. In this way miRNAs are able to repress gene expression post-transcriptionally by affecting mRNA stability or translation. These ubiquitous molecules play central roles in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Within the context of the immune system, genetic studies have identified distinct roles for specific miRNAs in gene regulation during development, activation and maturation. Conversely, dysregulation of miRNA expression has been specifically correlated with cancer. This review outlines our current understanding of miRNA function in lymphocytes as it impacts expression of protein-coding genes in the context of proper development, as well as oncogenesis.

Identifying mRNA editing deaminase targets by RNA-Seq.

RNA editing deaminases act on a variety of targets in different organisms. A number of such enzymes have been shown to act on mRNA, with the resultant nucleotide changes modifying a transcript's information content. Though the deaminase activity of mRNA editing enzymes is readily demonstrated in vitro, identifying their physiological targets has proved challenging. Recent advances in ultra high-throughput sequencing technologies have allowed for whole transcriptome sequencing and expression profiling (RNA-Seq). We have developed a system to identify novel mRNA editing deamination targets based on comparative analysis of RNA-Seq data. The efficacy and utility of this approach is demonstrated for APOBEC1, a cytidine deaminase with a known and well-characterized mRNA editing target in the mammalian small intestine.

Transcriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in transcript 3' UTRs.

Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.

A role for autophagic protein beclin 1 early in lymphocyte development.

Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.

Diverse functions for DNA and RNA editing in the immune system.

Polynucleotide DNA and RNA editing enzymes alter nucleic acid sequences and can thereby modify encoded informational content. Two major families of polynucleotide editing enzymes, the AID/APOBEC cytidine deaminases (which catalyze the deamination of cytidine to uridine) and the adenosine deaminases acting on RNA (ADARs, which catalyze the deamination of adenosine to inosine), function in a variety of host defense mechanisms. These enzymes act in innate and adaptive immune pathways, with both host and pathogen targets. DNA editing by the cytidine deaminase AID mediates immunoglobulin somatic hypermutation and class switch recombination, providing the antibody response with the flexibility and diversity to defend against an almost limitless array of varied and rapidly adapting pathogenic challenges. Other cytidine deaminases (APOBEC3) restrict retroviral infection by editing viral retrogenomes. Adenosine deaminases (ADARs) shape innate immune responses by modifying host transcripts that encode immune effectors and their regulators. Here we review current knowledge of polynucleotide DNA and RNA editors with a focus on these and other functions they serve in the immune system.

MicroRNAs of the immune system: roles in inflammation and cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to complementary target mRNAs and either promoting their decay or inhibiting their translation. Most eukaryotic genomes studied encode miRNAs, which are processed from longer noncoding transcripts through pathways conserved from fungi to plants to animals. miRNAs are now understood to be key mediators of developmental transitions in a number of model organisms. With respect to the immune system, miRNAs affect all facets of immune system development, from hematopoiesis to activation in response to infection during both the innate and the adaptive immune response. At the same time, miRNA dysregulation is a central event in the development and pathophysiology of a number of cancers of the immune system. Here we will discuss our current understanding of this general regulatory mechanism, focusing on its involvement in inflammation and in oncogenesis.

Shhh! Silencing by microRNA-155.

Small RNAs mediate a diverse pot-pourri of post-transcriptional silencing mechanisms, ranging from 'classical' RNA interference (RNAi), to gene repression by microRNAs (miRNAs), to maintenance of genomic stability by repeat-associated small RNAs. Here, we review recent findings on the function of miR-155, particularly its roles in mammalian innate and adaptive immunity, viral infection and oncogenesis.

C to U editing at position 32 of the anticodon loop precedes tRNA 5' leader removal in trypanosomatids.

In all organisms, precursor tRNAs are processed into mature functional units by post-transcriptional changes. These involve 5' and 3' end trimming as well as the addition of a significant number of chemical modifications, including RNA editing. The only known example of non-organellar C to U editing of tRNAs occurs in trypanosomatids. In this system, editing at position 32 of the anticodon loop of tRNA(Thr)(AGU) stimulates, but is not required for, the subsequent formation of inosine at position 34. In the present work, we expand the number of C to U edited tRNAs to include all the threonyl tRNA isoacceptors. Notably, the absence of a naturally encoded adenosine, at position 34, in two of these isoacceptors demonstrates that A to I is not required for C to U editing. We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5' leader removal. Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

A mammalian microRNA expression atlas based on small RNA library sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.