Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases.
ABSTRACT: Double-stranded (ds) RNA-specific adenosine deaminase converts adenosine residues into inosines in dsRNA and edits transcripts of certain cellular and viral genes such as glutamate receptor (GluR) subunits and hepatitis delta antigen. The first member of this type of deaminase, DRADA1, has been recently cloned based on the amino acid sequence information derived from biochemically purified proteins. Our search for DRADA1-like genes through expressed sequence tag databases led to the cloning of the second member of this class of enzyme, DRADA2, which has a high degree of sequence homology to DRADA1 yet exhibits a distinctive RNA editing site selectivity. There are four differentially spliced isoforms of human DRADA2. These different isoforms of recombinant DRADA2 proteins, including one which is a human homolog of the recently reported rat RED1, were analyzed in vitro for their GluR B subunit (GluR-B) RNA editing site selectivity. As originally reported for rat RED1, the DRADA2a and -2b isoforms edit GluR-B RNA efficiently at the so-called Q/R site, whereas DRADA1 barely edits this site. In contrast, the R/G site of GluR-B RNA was edited efficiently by the DRADA2a and -2b isoforms as well as DRADA1. Isoforms DRADA2c and -2d, which have a distinctive truncated shorter C-terminal structure, displayed weak adenosine-to-inosine conversion activity but no editing activity tested at three known sites of GluR-B RNA. The possible role of these DRADA2c and -2d isoforms in the regulatory mechanism of RNA editing is discussed.
Project description:The glutamate receptor subunit B (GluR-B) pre-mRNA is edited at two adenosine residues, resulting in amino acid changes that alter the electrophysiologic properties of the glutamate receptor. Previous studies showed that these amino acid changes are due to adenosine to inosine conversions in two codons resulting from adenosine deamination. Here, we describe the purification and characterization of an activity from human HeLa cells that efficiently and accurately edits GluR-B pre-mRNA at both of these sites. The purified activity contains a human homolog of the recently reported rat RED1 (rRED1) protein, a member of the family of double-stranded RNA-dependent deaminase proteins. Recombinant human RED1 (hRED1), but not recombinant dsRAD, another member of the family, efficiently edits both the Q/R and R/G sites of GluR-B RNA. We conclude that the GluR-B editing activity present in HeLa cell extracts and the recombinant hRED1 protein are indistinguishable.
Project description:We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.
Project description:RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.
Project description:RNA editing catalyzed by ADAR1 and ADAR2 involves the site-specific conversion of adenosine to inosine within imperfectly duplexed RNA. ADAR1- and ADAR2-mediated editing occurs within transcripts of glutamate receptors (GluR) in the brain and in hepatitis delta virus (HDV) RNA in the liver. Although the Q/R site within the GluR-B premessage is edited more efficiently by ADAR2 than it is by ADAR1, the converse is true for the +60 site within this same transcript. ADAR1 and ADAR2 are homologs having two common functional regions, an N-terminal double-stranded RNA-binding domain and a C-terminal deaminase domain. It is neither understood why only certain adenosines within a substrate molecule serve as targets for ADARs, nor is it known which domain of an ADAR confers its specificity for particular editing sites. To assess the importance of several aspects of RNA sequence and structure on editing, we evaluated 20 different mutated substrates, derived from four editing sites, for their ability to be edited by either ADAR1 or ADAR2. We found that when these derivatives contained an A:C mismatch at the editing site, editing by both ADARs was enhanced compared to when A:A or A:G mismatches or A:U base pairs occurred at the same site. Hence substrate recognition and/or catalysis by ADARs could involve the base that opposes the edited adenosine. In addition, by using protein chimeras in which the deaminase domains were exchanged between ADAR1 and ADAR2, we found that this domain played a dominant role in defining the substrate specificity of the resulting enzyme.
Project description:Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.
Project description:Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.
Project description:Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.
Project description:RNA editing generates protein diversity by altering RNA sequences in coding regions without changing the overall DNA sequence. Adenosine-to-inosine (A-to-I) RNA editing events have recently been reported in some types of cancer, but they are rare in human colorectal cancer (CRC). Therefore, this study was conducted to identify diverse RNA editing in CRC.We compared transcriptome data of 39 CRC samples and paired adjacent tissues from The Cancer Genome Atlas database to identify RNA editing patterns in CRC, focusing on canonical A-to-I RNA edits in coding sequence regions. We investigated nonsynonymous RNA editing patterns by comparing tumor and normal tissue transcriptome data.The number of RNA edits varied from 12 to 42 per sample. We also observed that hypoand hyper-RNA editing patterns were distinguishable within the samples. We found 10 recurrent nonsynonymous RNA editing candidates in nine genes (PDLIM, NEIL1, SRP9, GLI1, APMAP, IGFBP7, ZNF358, COPA, and ZNF587B) and validated some by Sanger sequencing and the inosine chemical erasing assay. We further showed that editing at these positions was performed by the adenosine deaminase acting on RNA 1 enzyme. Most of these genes are hypoedited in CRC, but editing of GLI1 was increased in cancer tissues compared with normal tissues.Our results show that nonsynonymous RNA editing patterns can be used to identify CRC patients and could serve as novel biomarkers for CRC.
Project description: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.
Project description:RNA editing by the host RNA adenosine deaminase ADAR1 at the amber/W site of hepatitis delta virus RNA plays a central role in the viral replication cycle by affecting the balance between viral RNA synthesis and packaging. Previously, we found that HDV genotype III (HDV-3) RNA can form two secondary structures following transcription: an unbranched rod structure, which is characteristic of HDV, and a metastable branched structure that serves as the substrate for editing. The unstable nature of the branched editing substrate structure raised the possibility that structural dynamics of the RNA following transcription could determine the rate at which editing occurs. Here, editing and its control are examined in two HDV-3 isolates, from Peru and Ecuador. Analysis of editing in vitro by ADAR1 indicated that the branched structure formed by RNA derived from the Peruvian isolate is edited more efficiently than that from the Ecuadorian isolate. In contrast, in the context of replication, Peruvian RNA is edited less efficiently than RNA containing Ecuadorian sequences. Computational analyses of RNA folding using the massively parallel genetic algorithm (MPGAfold) indicated that the Peruvian RNA is less likely to form the branched structure required for editing than the Ecuadorian isolate. This difference was confirmed by in vitro transcription of these RNAs. Overall, our data indicate that HDV-3 controls RNA editing levels via (1) the fraction of the RNA that folds, during transcription, into the metastable branched structure required for editing and (2) the efficiency with which ADAR1 edits this branched substrate RNA.