Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation ...pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.
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► The 50 kDa NMR structure of the two dsRBMs of ADAR2 bound to a 71 nt RNA ► Shape and sequence recognition of dsRNA by ADAR2 dsRBMs ► Importance of base-specific contacts for accurate substrate selection by ADAR2 ► Other dsRBMs may operate as sequence-specific RNA binding modules
The central nervous system-specific serotonin receptor 2C (5HT2C) controls key physiological functions, such as food intake, anxiety, and motoneuron activity. Its deregulation is involved in ...depression, suicidal behavior, and spasticity, making it the target for antipsychotic drugs, appetite controlling substances, and possibly anti-spasm agents. Through alternative pre-mRNA splicing and RNA editing, the 5HT2C gene generates at least 33 mRNA isoforms encoding 25 proteins. The 5HT2C is a G-protein coupled receptor that signals through phospholipase C, influencing the expression of immediate/early genes like c-fos. Most 5HT2C isoforms show constitutive activity, i.e., signal without ligand binding. The constitutive activity of 5HT2C is decreased by pre-mRNA editing as well as alternative pre-mRNA splicing, which generates a truncated isoform that switches off 5HT2C receptor activity through heterodimerization; showing that RNA processing regulates the constitutive activity of the 5HT2C system. RNA processing events influencing the constitutive activity target exon Vb that forms a stable double stranded RNA structure with its downstream intron. This structure can be targeted by small molecules and oligonucleotides that change exon Vb alternative splicing and influence 5HT2C signaling in mouse models, leading to a reduction in food intake. Thus, the 5HT2C system is a candidate for RNA therapy in multiple models of CNS disorders.
RNA editing can be broadly defined as any site-specific alteration in an RNA
sequence that could have been copied from the template, excluding changes due
to processes such as RNA splicing and ...polyadenylation. Changes in gene
expression attributed to editing have been described in organisms from
unicellular protozoa to man, and can affect the mRNAs, tRNAs, and rRNAs present
in all cellular compartments. These sequence revisions, which include both the
insertion and deletion of nucleotides, and the conversion of one base to
another, involve a wide range of largely unrelated mechanisms. Recent advances
in the development of in vitro editing and transgenic systems for these varied
modifications have provided a better understanding of similarities and
differences between the biochemical strategies, regulatory sequences, and
cellular factors responsible for such RNA processing events.
The enzyme ADAR2 is a double-stranded RNA-specific adenosine deaminase which is involved in the editing of mammalian messenger RNAs by the site-specific conversion of adenosine to inosine. Here we ...identify several rat ADAR2 mRNAs produced as a result of two distinct alternative splicing events. One such splicing event uses a proximal 3′ acceptor site, adding 47 nucleotides to the ADAR2 coding region, changing the predicted reading frame of the mature ADAR2 transcript. Nucleotide-sequence analysis of ADAR2 genomic DNA revealed the presence of adenosine-adenosine (AA) and adenosine-guanosine (AG) dinucleotides at these proximal and distal alternative 3′ acceptor sites, respectively. Use of the proximal 3′ acceptor depends upon the ability of ADAR2 to edit its own pre-mRNA, converting the intronic AA to an adenosine-inosine (Al) dinucleotide which effectively mimics the highly conserved AG sequence normally found at 3′ splice junctions. Our observations indicate that RNA editing can serve as a mechanism for regulating alternative splicing and they suggest a novel strategy by which ADAR2 can modulate its own expression.
The conversion of adenosine to inosine (A to I) by RNA editing represents a common posttranscriptional mechanism for diversification of both the transcriptome and proteome, and is a part of the ...cellular response for innate immune tolerance. Due to its preferential base-pairing with cytosine (C), inosine (I) is recognized as guanosine (G) by reverse transcriptase, as well as the cellular splicing and translation machinery. A-to-I editing events appear as A-G discrepancies between genomic DNA and cDNA sequences. Molecular analyses of RNA editing have leveraged these nucleoside differences to quantify RNA editing in ensemble populations of RNA transcripts and within individual cDNAs using high-throughput sequencing or Sanger sequencing-derived analysis of electropherogram peak heights. Here, we briefly review and compare these methods of RNA editing quantification, as well as provide experimental protocols by which such analyses may be achieved.
Calcium‐dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding ...CAPS1 are subject to a site‐specific adenosine‐to‐inosine (A‐to‐I) editing event resulting in a glutamate‐to‐glycine (E‐to‐G) substitution in the C‐terminal domain of the encoded protein product. The C‐terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non‐edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short‐term synaptic plasticity, specifically short‐term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non‐edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild‐type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.
Sole expression of the Calcium‐dependent activator protein for secretion 1 (CAPS1) isoform encoded by edited Cadps transcripts CAPS1(G) results in changes in short‐term plasticity and spontaneous neurotransmission at inhibitory synapses in primary hippocampal cultures without impacting excitatory transmission. CAPS1 is found to be expressed in approximately 85% and 5% of inhibitory and excitatory synapses, respectively, yet editing enhances synaptic localization of the protein to both terminal types. RNA editing of Cadps transcripts and adenosine deaminase acting on RNA (ADAR2) expression are modulated by neuronal activity.
Abstract
The hydrolytic deamination of adenosine-to-inosine (A-to-I) by RNA editing is a widespread post-transcriptional modification catalyzed by the adenosine deaminase acting on RNA (ADAR) family ...of proteins. ADAR-mediated RNA editing modulates cellular pathways involved in innate immunity, RNA splicing, RNA interference, and protein recoding, and has been investigated as a strategy for therapeutic intervention of genetic disorders. Despite advances in basic and translational research, the mechanisms regulating RNA editing are poorly understood. Though several trans-acting regulators of editing have been shown to modulate ADAR protein expression, previous studies have not identified factors that modulate ADAR catalytic activity. Here, we show that RNA editing increases upon intracellular acidification, and that these effects are predominantly explained by both enhanced ADAR base-flipping and deamination rate at acidic pH. We also show that the extent of RNA editing increases with the reduction in pH associated with conditions of cellular hypoxia.
Graphical Abstract
Graphical Abstract
Intracellular acidification drives protonation of a conserved glutamate in the ADAR deaminase domain, which in turn enhances base-flipping and increases the extent of RNA editing.
Key metabolic hormones, such as insulin, leptin, and adiponectin, have been studied extensively in obesity, however the pathophysiologic relevance of the calcitonin family of peptides remains ...unclear. This family includes calcitonin (CT), its precursor procalcitonin (PCT), and alpha calcitonin-gene related peptide (αCGRP), which are all encoded by the gene Calca. Here, we studied the role of Calca-derived peptides in diet-induced obesity (DIO) by challenging Calcr-/- (encoding the calcitonin receptor, CTR), Calca-/-, and αCGRP-/- mice and their respective littermates with high-fat diet (HFD) feeding for 16 weeks. HFD-induced pathologies were assessed by glucose tolerance, plasma cytokine and lipid markers, expression studies and histology. We found that DIO in mice lacking the CTR resulted in impaired glucose tolerance, features of enhanced nonalcoholic steatohepatitis (NASH) and adipose tissue inflammation compared to wildtype littermates. Furthermore, CTR-deficient mice were characterized by dyslipidemia and elevated HDL levels. In contrast, mice lacking Calca were protected from DIO, NASH and adipose tissue inflammation, and displayed improved glucose tolerance. Mice exclusively lacking αCGRP displayed a significantly less improved DIO phenotype compared to Calca-deficient mice. In summary, we demonstrate that the CT/CTR axis is involved in regulating plasma cholesterol levels while Calca, presumably through PCT, seems to have a detrimental effect in the context of metabolic disease. Our study provides the first comparative analyses of the roles of Calca-derived peptides and the CTR in metabolic disease.
Abstract RNA transcripts encoding the 2C-subtype of serotonin (5HT2C ) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects ...of altered 5HT2C editing in vivo , we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader–Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT2C receptor expression and 5HT2C -mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT2C gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT2C expression, but also demonstrate the importance of normal patterns of 5HT2C RNA editing in vivo.
The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding ...potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.
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