Extensive adenosine-to-inosine editing detected in Alu repeats of antisense RNAs reveals scarcity of sense-antisense duplex formation

Abstract

One type of RNA editing converts adenosine residues to inosine in double-stranded regions. Recent transcriptome analysis has revealed that numerous Alu repeats, present within introns and untranslated regions of human transcripts, are subject to this A-->I RNA editing. Furthermore, it revealed global transcription of antisense RNAs. Here, we demonstrate that antisense RNAs are also edited extensively but only in their Alu repeat sequences, and editing does not extend to the surrounding sequence. Our findings imply that sense and antisense RNAs form two separate intramolecular double-stranded RNAs consisting of inversely oriented Alu repeats, but rarely form intermolecular duplexes.

Fig. 1

Sense and antisense Alu editing in intron 2 of CNNM3. (A) Two inverted Alu repeats present in intron 2 of CNNM3 are shown as red and blue arrows. Antisense RNA corresponding to this region detected in this study is indicated also. The Alu repeats of the antisense RNA are presented as green and pink arrows. The small arrows indicate the RT-PCR primers, their directions, and their relative positions. The bold line with inverted arrows encompasses the region that is transcribed to prepare sense and antisense CNNM3 intron 2 RNAs examined for in vitro RNA editing assay. (B) A → I editing of Alu repeats. The editing frequency (%) at individual sites identified within the entire Alu Sg (262 bp; upper panel) and Alu Jb sequences (309 bp; lower panel) in sense (upper side) and antisense (lower side) RNA is summarized. (C) The secondary structure between two inverted Alu repeats in sense (upper) and antisense (lower) RNA is calculated by MFOLD. Highly edited sites (>30%) are indicated by vertical solid arrows. The blue arrow marked*: the site detected in vivo but not edited in vitro by recombinant ADAR proteins (see Fig. 3).

Fig. 2

Sense and antisense Alu editing in intron 16 of NFκB1. (A) Two inverted Alu repeats present in intron 16 of NFκB1 are shown as red and blue arrows. Antisense RNA corresponding to this region detected in this study is indicated also. The Alu repeats of the antisense RNA are presented as green and pink arrows. The small arrows indicate the RT-PCR primers, their directions, and their relative positions. (B) A → I editing of Alu repeats. The editing frequency (%) at individual sites identified within the entire Alu Y (306 bp; left panel) and Alu Sg sequences (290 bp; right panel) in sense (upper side) and antisense (lower side) RNA is summarized. (C) The secondary structure between two inverted Alu repeats in sense (upper) and antisense (lower) RNA is calculated by MFOLD. Highly edited sites (>30%) are indicated by vertical solid arrows.

Fig. 3

In vitro editing of sense and antisense CNNM3 intron 2 RNAs by ADARs. Highly edited sites (>30%) of sense (upper) and antisense (lower) CNNM3 intron 2 RNAs, which were mixed together (0.02 nM) and subjected to in vitro editing with recombinant ADAR1p110 (A) or ADAR2 (B), are indicated by vertical solid arrows. Essentially identical results were obtained at the RNA concentration of 0.2 nM (data not shown).