A method to find tissue-specific novel sites of selective adenosine deamination

Ohlson, Johan; Ensterö, Mats; Sjöberg, Britt‐Marie; Öhman, Marie

doi:10.1093/nar/gni169

Cited by 29 publications

(25 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have developed a method by which it is possible to find novel ADAR substrates that have been edited in a siteselective manner (Ohlson et al 2005). The method is based on extracting intrinsic ADAR2-RNA substrate complexes by coimmunoprecipitation using an anti-ADAR2 antibody.…”

Section: Detecting Novel Sites Of A-to-i Editingmentioning

confidence: 99%

See 1 more Smart Citation

Editing modifies the GABAA receptor subunit 3

Ohlson¹,

Pedersen²,

Haussler³

et al. 2007

RNA

Self Cite

185

175

View full text Add to dashboard Cite

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the a3 subunit of the GABA A receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.

show abstract

Section: Detecting Novel Sites Of A-to-i Editingmentioning

confidence: 99%

“…We have combined bioinformatic analyzes detecting stem-loop structures suitable for A-to-I editing (Pedersen et al 2006) with an experimental method developed to find novel ADAR substrates that have been edited in a siteselective manner (Ohlson et al 2005). Several potential editing substrates were discovered using this approach.…”

Section: Introductionmentioning

confidence: 99%

Editing modifies the GABAA receptor subunit 3

Ohlson¹,

Pedersen²,

Haussler³

et al. 2007

RNA

Self Cite

185

175

View full text Add to dashboard Cite

show abstract

“…Although A-to-I editing clearly has important consequences in vertebrates, available data would suggest that it is infrequently used to modify protein structure. In spite of numerous mammalian genome sequences, EST and SNP databases, fewer than 30 mRNAs have been found to be edited in their coding sequence, at an average of z1.8 sites per transcript (Clutterbuck et al 2005;Levanon et al 2005;Ohlson et al 2005;Gommans et al 2008).…”

Section: Introductionmentioning

confidence: 99%

An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme

Palavicini¹,

O’Connell²,

Rosenthal³

2009

RNA

View full text Add to dashboard Cite

RNA editing by adenosine deamination is particularly prevalent in the squid nervous system. We hypothesized that the squid editing enzyme might contain structural differences that help explain this phenomenon. As a first step, a squid adenosine deaminase that acts on RNA (sqADAR2a) cDNA and the gene that encodes it were cloned from the giant axon system. PCR and RNase protection assays showed that a splice variant of this clone (sqADAR2b) was also expressed in this tissue. Both versions are homologous to the vertebrate ADAR2 family. sqADAR2b encodes a conventional ADAR2 family member with an evolutionarily conserved deaminase domain and two double-stranded RNA binding domains (dsRBD). sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an ''extra'' dsRBD. Both splice variants are expressed at comparable levels and are extensively edited, each in a unique pattern. Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. Using a standard 48-h protein induction, both sqADAR2a and sqADAR2b exhibit promiscuous self-editing; however, this activity is particularly robust for sqADAR2a. By decreasing the induction time to 16 h, self-editing was mostly eliminated. We next tested the ability of sqADAR2a and sqADAR2b to edit two K + channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. These data suggest that the ''extra'' dsRBD confers high activity on sqADAR2a.

show abstract

“…The glutamate receptor Gria2, the serotonin receptor Htr2C, the potassium voltage-gated channel Kcna1, and the γ-aminobutyric acid receptor Gabra3 are reported as targets of the Adar proteins, and are all highly expressed in the mouse brain (Sommer et al 1991;Higuchi et al 1993;Burns et al 1997;Hoopengardner et al 2003;Bhalla et al 2004;Daniel et al 2010). Enrichment of these transcripts was compared with the relative enrichment of a set of negative controls, ß-Actin, Gapdh, Ppia, and Atp5e, which have no annotated editing sites in the DARNED database (Kiran and Baranov 2010), and Rplp0, which has been used as a negative control in previous A > I editing experiments (Ohlson et al 2005;. Gabra3, Htr2C, Gria2, and Kcna1 showed three-to sixfold enrichment in the I-RNA pool, whereas the levels of Atp5e, Ppia, Rplp0, and Gapdh were relatively depleted, demonstrating that the protocol facilitated an efficient enrichment of edited RNA species ( Fig.…”

Section: Specific Enrichment Of Known Adar Targetsmentioning

confidence: 99%

“…For example, Ohlson and colleagues performed ADAR immunoprecipitations and identified the ADAR-associated RNAs by microarray. These results, however, were not only restricted to the transcripts with probes on the microarray, but also limited due to the fact that a transcript's physical association with ADAR is not necessarily evidence of editing (Ohlson et al 2005;. More recently, Tseng et al (2013) developed a method to detect RNA containing inosine by microarray and Sakurai and colleagues (Sakurai et al 2010;Sakurai and Suzuki 2011) developed a protocol in which they used inosine cyanoethylation to block reverse transcription, which therefore allowed them to compare treated and untreated cDNAs to identify putative editing sites.…”

Section: Introductionmentioning

confidence: 95%

Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage

Cattenoz¹,

Taft²,

Westhof³

et al. 2012

RNA

View full text Add to dashboard Cite

Adenosine to inosine (A > I) RNA editing, which is catalyzed by the ADAR family of proteins, is one of the fundamental mechanisms by which transcriptomic diversity is generated. Indeed, a number of genome-wide analyses have shown that A > I editing is not limited to a few mRNAs, as originally thought, but occurs widely across the transcriptome, especially in the brain. Importantly, there is increasing evidence that A > I editing is essential for animal development and nervous system function. To more efficiently characterize the complete catalog of ADAR events in the mammalian transcriptome we developed a high-throughput protocol to identify A > I editing sites, which exploits the capacity of glyoxal to protect guanosine, but not inosine, from RNAse T1 treatment, thus facilitating extraction of RNA fragments with inosine bases at their termini for high-throughput sequencing. Using this method we identified 665 editing sites in mouse brain RNA, including most known sites and suite of novel sites that include nonsynonymous changes to protein-coding genes, hyperediting of genes known to regulate p53, and alterations to non-protein-coding RNAs. This method is applicable to any biological system for the de novo discovery of A > I editing sites, and avoids the complicated informatic and practical issues associated with editing site identification using traditional RNA sequencing data. This approach has the potential to substantially increase our understanding of the extent and function of RNA editing, and thereby to shed light on the role of transcriptional plasticity in evolution, development, and cognition.

show abstract

A method to find tissue-specific novel sites of selective adenosine deamination

Cited by 29 publications

References 30 publications

Editing modifies the GABAA receptor subunit 3

Editing modifies the GABAA receptor subunit 3

An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme

Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage

Contact Info

Product

Resources

About