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.

Keywords: ADAR; Adenosine deamination; EIE; Glutamate receptor; RNA editing; miRNA.

Fig. 1

Structural requirements for efficient editing at the Q/R site of the GluA2 transcript. a GluA2 RNA structure at exon11–intron11. Exonic sequence is illustrated in blue and intronic sequence in black. The Q/R site is located in exon 11 and indicated with a red dot. The region in grey illustrates the position of the EIE, 45 nt downstream of the Q/R site. b Left: the wild-type construct, GA2Q/R containing the Q/R editing site and the EIE; the GA2Q/R-ΔEIE mutant where the EIE has been deleted; the GA2Q/R-US EIE where the EIE has been moved to a position 50 nt upstream of the Q/R site; and GA2Q/R-US G3 EIE where the Gabra-3 EIE is placed 50 nt upstream of the Q/R site. Right: sequencing chromatograms illustrating editing of the different GluA2 reporters by endogenous ADAR2 in HeLa cells. c Quantification of editing efficiency at the Q/R site from the different GA2Q/R constructs in HeLa cells. The mean value of the ratio between the A and G peak heights from three individual experiments is calculated as the percentage of editing. Error bars are standard deviation

Fig. 2

The EIE of GluA2 can induce editing at the IM site in Gabra-3. a The wild-type Gabra-3 construct (G3 I/M) showing the short stem structure formed at the edited I/M site (red dot) and the EIE (in grey); the G3 I/M-ΔEIE mutant were the EIE has been deleted; and G3 I/M-DS GA2 EIE were the Gabra-3 EIE is replaced by the GluA2 EIE. b Quantification of editing efficiency at the I/M site in the different G3I/M constructs transfected into HeLa cells. c Quantification of editing efficiency at I/M site from the different G3I/M constructs when co-transfected with ADAR1 or ADAR2 in HEK293 cells. The mean value of the ratio between the A and G peak heights from three separate experiments was calculated as percentage editing. Error bars are standard deviation

Fig. 3

Selectivity of editing at the Q/R site in GluA2. a Top: sites of editing and average percentage editing in the GluA2 reporter GA2Q/R co-transfected with an ADAR2 expression vector in HEK293 cells. Exon sequence is indicated in blue and the editing inducer element (EIE) shaded in grey. The Q/R site is indicated with a red dot. Bottom: sites of editing in the GluA2 reporter with the internal loop deleted (GA2Q/R-Δloop) co-transfected with ADAR2 in HEK293 cells. b Top: sites of editing and average percentage editing in the GluA2 reporter GA2Q/R co-transfected with the mutant ADAR2-EAA-E488Q expression vector in HEK293 cells. Bottom: sites of editing in the GluA2 reporter with the internal loop deleted (GA2Q/R-Δloop) co-transfected with ADAR2-EAA-E488Q in HEK293 cells. The mean value of the ratio between the A and G peak heights from three separate experiments was calculated as percentage editing

Fig. 4

Structural requirements for efficient editing at the Q/R site in the GluK2 transcript. a Top: the GK2Q/R construct showing the structure formed in the vicinity of the Q/R site. The edited adenosine (red dot) is located in exon 12 and pairing sequences are located within intron 12. Three stems—the Upstream stem, Q/R stem, and Downstream stem—are separated by larger internal loops. The region in grey illustrates the position of the EIE. Middle: the GK2Q/R-ΔEIE USS reporter where the upstream stem has been deleted. Bottom: the GK2Q/R-ΔEIE DSS reporter where the downstream stem has been disrupted. b Quantification of editing efficiency at the Q/R site from the different GK2Q/R constructs transfected into HeLa cells. c Quantification of editing efficiency at the Q/R site from the different GK2Q/R constructs co-transfected with ADAR2 in HEK 293 cells. The mean value of the ratio between the A and G peak heights from three individual experiments was calculated as the percentage of editing. Error bars are standard deviation

Fig. 5

Editing at the I/V site of Kv1.1 is induced by an EIE. a Left: mfold structure prediction of the Kv1.1 transcript in the vicinity of the I/V site. The blue arrow indicates the I/V site. Right: the three Kv1.1 I/V editing reporter constructs illustrating the insertion of the EIE from GluA2 (kv1.1-Q/R EIE), the insertion of the EIE from Gabra-3 (Kv1.1-G3 EIE), and the insertion of the Gabra-3 EIE both upstream and downstream of the Kv1.1 stem loop. b Quantification of editing efficiency at the I/V site from the different Kv1.1 constructs transfected into HeLa cells, as indicated. c Quantification of editing efficiency at the I/V site from the different Kv1.1 constructs co-transfected with ADAR2 in HEK 293 cells. The mean value of the ratio between the A and G peak heights from three individual experiments was calculated as percentage of editing. Error bars are standard deviation

Fig. 6

Structural requirements for efficient editing at the 5′ +4 site and 3′ +6 sites of the pri-miR-376a2 transcript. a Top: the miR-376 cluster constructs. Edited adenosines in the pri-miRNA stem loops are indicated with red dots and efficiently edited adenosines are shown with numbers indicating their position in the corresponding mature miRNAs. Mature miRNA sequences are indicated with blue lines. Below: sequences of different pre-miR constructs from the miR-376 cluster. WT indicates the full length of the cluster shown above. b Quantification of editing efficiency at the 5′ +4 site and 3′ +6 sites of the pri-miR-376a2 in the different constructs transfected into HeLa cells. The mean value of the ratio between the A and G peak heights from three individual experiments was calculated as percentage of editing. Error bars are standard deviation

Fig. 7

A model for efficient site selective A-to-I editing using an editing inducer element (EIE). The process of efficient editing occurs as two consecutive events: 1) ADAR (in blue) recognizes a longer intronic stem by a non-specific interaction; 2) when the ADAR enzymes have been recruited, the catalytic domain of the protein interacts with a specific site, ideal for catalysis, situated in a shorter stem limited by a barbell-like structure (in grey). The site of selective editing is indicated in red