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.

Figure 1

(A) The method used to detect inosine-containing RNAs. As described (18), inosine-specific cleavage is followed by polyadenylation to create a primer binding site for a subsequent reverse transcription step. N, nucleotide 5′ of poly(A) tail; the question mark indicates that efficiency of synthesis from uncleaved RNAs will vary depending on the nucleotide 5′ of the poly(A) tail. X, nucleotide(s) on the 3′ end of the downstream PCR primer was G, A, C, TG, TA, TT, or TC. (B) An example of an RNase T1-dependent band. Duplicate samples of human poly(A)⁺ RNA were subjected to the protocol of A. The arrow points to the RNase T1-dependent band (in the + lanes) whose sequence revealed that it derived from HsC7-I mRNA cleaved in its 3′ UTR.

Figure 2

Each of the newly identified C. elegans substrates is designated with a name, the location of editing within the mRNA, and the type of repetitive element, if one exists. Structures predicted by mfold are shown above the unedited sequence. Adenosines determined to be edited by cDNA sequencing are shown in uppercase, color-coded according to the percentage of the cDNA population that was edited at the site (red, >70%; blue, 40–70%; green, <40%); RNase T1 cleavage sites are underlined. Editing in syntaxin occurs within an alternatively spliced region that can be either an intron or a 3′ UTR. SSADH was found by virtue of its IR element, not by T1 cleavage, and only nucleotides 2538–2773 (complementary strand) of cosmid F45H10 were sequenced to determine editing sites. Editing sites were mapped by compiling sequence data from multiple PCR clones (SSADH), uncloned PCR populations (36A), or both types of data (syntaxin; laminin-γ); the editing sites of 12A have not been mapped, but three T1 cleavage sites were detected and are shown as black underlined capitals.

Figure 3

(A) The electropherograms correspond to the first two editing sites of HsC7-1 mRNA. The two adenosines shown in the genomic DNA sequence appear as a mixture of adenosine and guanosine in the cDNA, which is diagnostic of editing. N indicates that the sequencing software could not distinguish between A and G. (B) Eight of the newly identified human brain substrates are represented as in Fig. 2. (The RNase T1 cleavage site in the CDK8 mRNA was on the bottom strand, which was not sequenced.)