RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA

Abstract

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. We previously developed a method to identify inosine-containing RNAs and used it to identify five ADAR substrates in Caenorhabditis elegans. Here we use the same method to identify five additional C. elegans substrates, including three mRNAs that encode proteins known to affect neuronal functions. All 10 of the C. elegans substrates are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions was a conserved ADAR function, we applied our method to poly(A) ؉ RNA of human brain and identified 19 previously unknown ADAR substrates. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3 untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule.A denosine deaminases that act on RNA (ADARs) are RNAediting enzymes that convert adenosine (A) to inosine (I) in double-stranded RNA (dsRNA) (reviewed in refs. 1-4). Mice (5, 6), flies (7), and worms (L. A. Tonkin, L. Saccomanno, D.P.M., T. Brodigan, M. Krause, and B.L.B., unpublished work) that lack ADARs have behavioral defects, emphasizing that ADARs have important roles in the nervous system. However, exactly how the enzymes mediate their functions, and which RNAs they target, is known for only a few cases. For example, in mammals ADARs are known to target codons so that multiple protein isoforms can be synthesized from a single message; in this way ADARs generate isoforms of glutamate receptors (GluRs) (8-10), serotonin receptors (11), and the hepatitis delta antigen (12, 13). In addition, mammalian ADAR2 edits its own pre-mRNA to create a new splice site, which may serve to autoregulate ADAR2 levels (14). Finally, recent data show that RNAs targeted by ADARs at many sites are selectively retained in the nucleus (15), at least in some cases.In addition to altering the sequence of RNA, ADARs can alter RNA structure. Deamination of an adenosine within an A⅐U base pair produces the less stable I⅐U wobble pair, whereas deamination within A⅐C mismatches produces more stable I⅐C pairs. Therefore, ADARs can either decrease or increase the stability of dsRNA. Sequence or structural changes produced by ADARs within noncoding regions of mRNAs such as untranslated regions (UTRs), or introns, could potentially affect such processes as RNA translation, stability, localization, or splicing. Other types of RNAs such as catalytic RNAs, guide RNAs, small nuclear RNAs, small nucleolar RNAs...

Widespread RNA Editing of Embedded Alu Elements in the Human Transcriptome

“…The editing targets identified in the current study may represent a small portion of this "hidden" transcriptome. Consistent with this idea, only two of the 15 previously found Alu-associated editing targets (Morse et al 2002) overlapped with our selected transcripts. dsRNAs longer than 30 bp induce dsRNA-dependent pathways in mammalian cells such as the interferon response, with activation of PKR and 2Ј,5Ј-oligoadenylate (Samuel 2001).…”

“…Although it is conceivable that targeting is based on particular nucleotide motifs intrinsic to Alu sequences, considering the nature of ADARs to recognize and bind any dsRNA structure, an a priori explanation is the capacity for Alus within a primary transcript to form extended RNA duplexes (Kikuno et al 2002;Morse et al 2002). The enormous number of Alus present in either orientation within transcribed regions makes the intramolecular formation of dsRNA by inverted Alu repeats the most plausible scenario, although dsRNA formation in trans is also possible.…”

“…Non-repeats refer to sequences other than interspersed complex repeats and include unique sequences as well as low complexity repeats and satellites. repeat sequences, as well as the experimental observation of inosine within Alus in some brain transcripts (Morse et al 2002), we believe that the selected transcripts represent genuinely edited RNAs. Other explanations for the overabundance of targeted A-to G substitutions are implausible.…”

“…In contrast, multiple A-to-I modifications are observed in some viral RNAs, particularly the hypermutations found in certain minus-strand virus genomes. A recent method developed to find edited RNA unexpectedly revealed 19 transcripts from human brain with multiple edited bases in noncoding regions, raising the possibility that this type of editing might be more common than single base editing within coding regions (Morse et al 2002). Interestingly, 15 of those 19 transcripts were edited within repetitive elements present in introns or UTRs.…”

Large-Scale Overexpression and Purification of ADARs from Saccharomyces cerevisiae for Biophysical and Biochemical Studies

“…This is supported by evidence showing that knockouts of ADARs lead to various behavioral abnormalities (Higuchi et al, 2000;Palladino et al, 2000a;Tonkin et al, 2002) and that several known pre-mRNA substrates code for neuronal receptors and ion channels (Burns et al, 1997;Higuchi et al, 1993;Lomeli et al, 1994). Though the effects of editing coding sequences are dramatic, these editing events are rare relative to the levels of editing in noncoding regions, especially untranslated regions (UTRs) (Blow et al, 2004;Levanon et al, 2004;Morse and Bass, 1999;Morse et al, 2002). The extensive editing of noncoding regions has been proposed to affect expression of a gene posttranscriptionally, although how this might occur is unclear.…”