Recognition and coupling of A-to-I edited sites are determined by the tertiary structure of the RNA

Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate

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

Abstract 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

Large-scale mRNA sequencing determines global regulation of RNA editing during brain development

RNA editing by adenosine deamination has been shown to generate multiple isoforms of several neural receptors, often with profound effects on receptor function. However, little is known about the regulation of editing activity during development. We have developed a large-scale RNA sequencing protocol to determine adenosine-to-inosine (A-to-I) editing frequencies in the coding region of genes in the mammalian brain. Using the 454 Life Sciences (Roche) Amplicon Sequencing technology, we were able to determine even low levels of editing with high accuracy. The efficiency of editing for 28 different sites was analyzed during the development of the mouse brain from embryogenesis to adulthood. We show that, with few exceptions, the editing efficiency is low during embryogenesis, increasing gradually at different rates up to the adult mouse. The variation in editing gave receptors like HTR2C and GABAA (gamma-aminobutyric acid type A) a different set of protein isoforms during development from those in the adult animal. Furthermore, we show that this regulation of editing activity cannot be explained by an altered expression of the ADAR proteins but, rather, by the presence of a regulatory network that controls the editing activity during development

A distant cis acting intronic element induces site-selective RNA editing

Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome.AuthorCount:5;</p

Additional file 1: Figure S1. of Editing inducer elements increases A-to-I editing efficiency in the mammalian transcriptome

Quantification of editing efficiency at the Q/R sit from the different GA2Q/R reporters cotransfected with ADAR2 in HEK 293 cells. The mean value of the ratio between the A and G peak heights from three individual experiments are calculated as percentage of editing. Error bars are standard deviation. The value of GA2Q/R-ΔEIE was significantly different to the values of all the other reporters, the values of the GA2Q/R-US EIE and GAQ/R-US G3 EIE where not significantly different to the WT GA2Q/R reporter (P=0.05 two tailed student’s ttest). Figure S2. Titration of ADAR2 co-transfected with GA2Q/R or GA2Q/R-ΔEIE. (a) Sequencing chromatograms of RT-PCR products from ADAR2 co-transfections with GA2Q/R or GA2Q/R-ΔEIE. In each experiment, transfection of the reporter constructs was constant (0.75μg), while the concentration of ADAR2 was titrated (0-1.25μg). (b) Quantification of the Q/R editing efficiency in GA2Q/R (dots) and GA2Q/R-ΔEIE (squares) reporters when co-transfected with titrated ADAR2. Three individual experiments were done for each concentration. The mean value of the ratio between the A and G peak heights was calculated as percentage of editing. Error bars are standard deviation. Figure S3. (a) Sites of editing and average % editing in the GluA2 reporter GA2Q/R cotransfected with the mutant ADAR2-E488Q expression vector in HEK293 cells. Below, sites of editing in the GluA2 reporter with the internal loop deleted (GA2Q/R-Δloop) co-transfected with ADAR2-E488Q in HEK293. The average value of the ratio between the A and G peak heights from two separate experiments was calculated as percentage editing. (b) Western blot showing expression levels of different transiently transfected ADAR2 expression vectors shown in A and Figure 3. EV equals transfection of empty vector as control. Figure S4. Predicted RNA secondary structure of the EIE in mouse GluA2 and GluK2. Differences in the human sequences are indicated by arrows and base changes in blue. Edited adenosines in the mouse sequence are shown in read. (PDF 3470 kb