Editing inducer elements increases A-to-I editing efficiency in the mammalian transcriptome

Genome Biology, Oct 2017

Adenosine to inosine (A-to-I) RNA editing has been shown to be an essential event that plays a significant role in neuronal function, as well as innate immunity, in mammals. It requires a structure that is largely double-stranded for catalysis but little is known about what determines editing efficiency and specificity in vivo. We have previously shown that some editing sites require adjacent long stem loop structures acting as editing inducer elements (EIEs) for efficient editing. The glutamate receptor subunit A2 is edited at the Q/R site in almost 100% of all transcripts. We show that efficient editing at the Q/R site requires an EIE in the downstream intron, separated by an internal loop. Also, other efficiently edited sites are flanked by conserved, highly structured EIEs and we propose that this is a general requisite for efficient editing, while sites with low levels of editing lack EIEs. This phenomenon is not limited to mRNA, as non-coding primary miRNAs also use EIEs to recruit ADAR to specific sites. We propose a model where two regions of dsRNA are required for efficient editing: first, an RNA stem that recruits ADAR and increases the local concentration of the enzyme, then a shorter, less stable duplex that is ideal for efficient and specific catalysis. This discovery changes the way we define and determine a substrate for A-to-I editing. This will be important in the discovery of novel editing sites, as well as explaining cases of altered editing in relation to disease.

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Editing inducer elements increases A-to-I editing efficiency in the mammalian transcriptome

Daniel et al. Genome Biology Editing inducer elements increases A-to-I editing efficiency in the mammalian transcriptome Chammiran Daniel 0 Albin Widmark 0 Ditte Rigardt 0 Marie Öhman 0 0 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , Svante Arrheniusväg 20C, 10691 Stockholm , Sweden Background: Adenosine to inosine (A-to-I) RNA editing has been shown to be an essential event that plays a significant role in neuronal function, as well as innate immunity, in mammals. It requires a structure that is largely double-stranded for catalysis but little is known about what determines editing efficiency and specificity in vivo. We have previously shown that some editing sites require adjacent long stem loop structures acting as editing inducer elements (EIEs) for efficient editing. Results: The glutamate receptor subunit A2 is edited at the Q/R site in almost 100% of all transcripts. We show that efficient editing at the Q/R site requires an EIE in the downstream intron, separated by an internal loop. Also, other efficiently edited sites are flanked by conserved, highly structured EIEs and we propose that this is a general requisite for efficient editing, while sites with low levels of editing lack EIEs. This phenomenon is not limited to mRNA, as non-coding primary miRNAs also use EIEs to recruit ADAR to specific sites. Conclusions: We propose a model where two regions of dsRNA are required for efficient editing: first, an RNA stem that recruits ADAR and increases the local concentration of the enzyme, then a shorter, less stable duplex that is ideal for efficient and specific catalysis. This discovery changes the way we define and determine a substrate for A-to-I editing. This will be important in the discovery of novel editing sites, as well as explaining cases of altered editing in relation to disease. RNA editing; Adenosine deamination; Glutamate receptor; ADAR; EIE; miRNA Background Complex organisms require a great diversity of gene products for proper development and function, particularly in the brain. This is achieved by the use of numerous co- or post-transcriptional processes, such as alternative splicing, alternative polyadenylation, and RNA editing. Adenosine-to-inosine (A-to-I) RNA editing is a highly conserved RNA modification process that occurs in all metazoan lineages [ 1 ]. Inosine base pairs with C and is interpreted as G by the cellular machineries. Hence, A-to-I RNA editing can be designated as an A-to-G conversion and, if situated in exonic sequence, it has the potential to alter codons and consequently contribute to the expression of additional protein isoforms (reviewed in [ 2 ]). A-to-I conversions within introns and 3′ UTRs can also have an impact on the transcriptome, e.g., by creating new splice sites and changing miRNA target recognition. A-to-I editing is essential to the organism and aberrant editing has been linked to a variety of different human diseases: amyotrophic lateral sclerosis (ALS) and other neurological disorders, several types of cancer, and autoimmune disorders such as the Aicardi-Goutières syndrome (AGS) [ 3–6 ]. To understand what determines the level of editing in different substrates and under different circumstances, we need to know the mechanism of substrate recognition. It is, however, still largely unclear what factors determine the efficiency of editing. A-to-I RNA editing is performed by the adenosine deaminases that act on RNA (ADAR) enzymes that recognize adenosines located in double-stranded RNA (dsRNA) to be deaminated into inosines [ 7 ]. ADAR proteins are evolutionarily conserved in metazoans and mammals have two enzymatically active ADAR enzymes, ADAR1 and ADAR2 [ 8–10 ]. In some cases, the substrate selectivity of the two enzymes overlaps, but more commonly the targets are specific for either enzyme [ 11–13 ]. ADAR1 and ADAR2 share certain domain structures, such as the deaminase domain and the double-stranded RNA binding domains (dsRBDs). However, the numbers of dsRBDs differ between the two enzymes (ADAR1 contains three while ADAR2 contains two) as well as the spacing between them. The dsRBDs recognize one face of the sugar backbone of an A-form helix, such as the RNA duplex, spanning two minor grooves and an intervening major groove [ 14 ]. Thus, there is little sequence specificity via interaction of the dsRBDs and theoretically they can interact with any double-stranded RNA longer than 16 nucleotides (nt). However, sequence-specific interactions between the two dsRBDs of human ADAR2 at the GluA2 stem loop at the R/G site have been reported based on the NMR structure [ 15 ]. Interestingly, it has recently been shown that the deaminase domain also requires a doublestranded structure in order to interact with the substrate and perform the catalysis [ 16, 17 ]. In general, there are two categories of A-to-I RNA editing determined by the structure of the RNA. Long double-stranded structu (...truncated)


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Chammiran Daniel, Albin Widmark, Ditte Rigardt, Marie Öhman. Editing inducer elements increases A-to-I editing efficiency in the mammalian transcriptome, Genome Biology, 2017, pp. 195,