Specificity of ADAR-mediated RNA editing in newly identified targets

Abstract

Adenosine deaminases that act on RNA (ADARs) convert adenosines to inosine in both coding and noncoding double-stranded RNA. Deficiency in either ADAR1 or ADAR2 in mice is incompatible with normal life and development. While the ADAR2 knockout phenotype can be attributed to the lack of editing of the GluR-B receptor, the embryonic lethal phenotype caused by ADAR1 deficiency still awaits clarification. Recently, massive editing was observed in noncoding regions of mRNAs in mice and humans. Moreover, editing was observed in protein-coding regions of four mRNAs encoding FlnA, CyFip2, Blcap, and IGFBP7. Here, we investigate which of the two active mammalian ADAR enzymes is responsible for editing of these RNAs and whether any of them could possibly contribute to the phenotype observed in ADAR knockout mice. Editing of Blcap, FlnA, and some sites within B1 and B2 SINEs clearly depends on ADAR1, while other sites depend on ADAR2. Based on our data, substrate specificities can be further defined for ADAR1 and ADAR2. Future studies on the biological implications associated with a changed editing status of the studied ADAR targets will tell whether one of them turns out to be directly or indirectly responsible for the severe phenotype caused by ADAR1 deficiency.

FIGURE 1.

Independent nearest-neighbor preferences. Occurrence of 5′and 3′ neighbors, as well as opposite bases were analyzed for all edited and unedited adenosines found in B1 and B2 elements. The frequency of bases neighboring the edited adenosines are indicated in blue-gray bubbles, while neighbors of unedited bases are shown in white bubbles. Strong deviation of the values between the blue and white bubbles are indicative of a sequence preference by ADARs. Each value is subdivided into sites edited exclusively by ADAR1 (indicated in light blue), ADAR2 (indicated in gray), or both enzymes (white). Opposite base preferences were only determined for adenosines clearly localized in well-base-paired regions.

FIGURE 2.

Triplet preferences. Frequencies of triplets are shown in percent (blue-gray bubbles). Each value is subdivided into sites edited exclusively by ADAR1 (indicated in light blue), ADAR2 (indicated in gray), or both enzymes (white). To take sequence bias into account, these values represent the frequency of edited triplets corrected against their overall occurrence within the dsRNA structures.

FIGURE 3.

Detail of a B1 alignment. B1 elements of Exoc8 and CD300a were aligned. A detail from this alignment highlighting the clustering of editing events in a defined region (boxed area in Supplemental Fig. 1) is shown. Editing of two positions (sites 2 and 4 in CD300a, and 6 and 7 in Exoc8) is conserved between the two examined B1 elements. Sites edited exclusively by ADAR1 or ADAR2 are indicated in blue or green, respectively. Sites edited by either enzyme are indicated in orange. Coordinates: from accession numbers NM_198103 for Exoc8, NM_170758 for CD300a; the first number indicates the local position where the first nucleotide of the first repeat = 1, second number indicates relative position of the repeat within the entire pre-mRNA transcript. The full alignment can be seen in the supplements.