Adenosine deamination in human transcripts generates novel microRNA binding sites

Abstract

Animals regulate gene expression at multiple levels, contributing to the complexity of the proteome. Among these regulatory events are post-transcriptional gene silencing, mediated by small non-coding RNAs (e.g. microRNAs), and adenosine-to-inosine (A-to-I) editing, generated by adenosine deaminases that act on double-stranded RNA (ADAR). Recent data suggest that these regulatory processes are connected at a fundamental level. A-to-I editing can affect Drosha processing or directly alter the microRNA (miRNA) sequences responsible for mRNA targeting. Here, we analyzed the previously reported adenosine deaminations occurring in human cDNAs, and asked if there was a relationship between A-to-I editing events in the mRNA 3' untranslated regions (UTRs) and mRNA:miRNA binding. We find significant correlations between A-to-I editing and changes in miRNA complementarities. In all, over 3000 of the 12 723 distinct adenosine deaminations assessed were found to form 7-mer complementarities (known as seed matches) to a subset of human miRNAs. In 200 of the ESTs, we also noted editing within a specific 13 nucleotide motif. Strikingly, deamination of this motif simultaneously creates seed matches to three (otherwise unrelated) miRNAs. Our results suggest the creation of miRNA regulatory sites as a novel function for ADAR activity. Consequently, many miRNA target sites may only be identifiable through examining expressed sequences.

Figure 1.

ADARs deaminate adenosine to inosine, potentially altering miRNA complementarities. (A) A cartoon depicting adenosine, deaminated adenosine (inosine) and guanine. (B) A representative deamination site occurring in the 3′-UTR of DNA fragmentation factor α (DFFA) is shown in both the unedited (left) and edited (right) state. For this transcript, the 7 nt sequence scan (red) of the 200 nts flanking the deamination site (blue) identified a miR-513 seed sequence (yellow). This was repeated for all miRNA seeds (miRBase 13.0) for all known edited transcripts.

Figure 2.

A-to-I edits create miR-513 and miR-769-3p/-450b-3p complementarities. (A) 12 723 unique EST sequences (www.cgen.com), each consisting of a central A-to-I deamination and 100 nt flanks [i.e. n₁₀₀ (A or I) n₁₀₀], were screened for complementarity to human miRNAs. All human miRNA seed matches were identified within the individual 201 nt sequences originally identified as an A-to-I transition by Compugen (statistical significance is addressed in Table 1). The top two panels represent all miR-769-3p (and miR-450b-3p) seed matches occurring at each position in both the unedited (left) and edited (right) states. The lower panels represent all miR-513 seed matches occurring in unedited (left) and edited (right) states. (B) A cartoon of miR-513 and miR-769-3p complementarities to a miRNA Associating If Deaminated (MAID) site in both unedited (left) and edited (right) state is shown. The seed of miR-450b-3p (not shown) is identical to miR-769-3p. Perfect seed matches to miR-769-3p/-450b-3p (blue) and miR-513 (yellow) are significantly enriched in sequences containing characterized deaminations (red). Vertical lines indicate complementary base pairing. (C) Venn diagram depicting the overlap between miR-513 and miR-769-3p/-450b-3p target sites matching the full 13-bp MAID motif created by adjacent miR-513 and miR-769/-450-3p seeds and a G:U wobble. Nearly 100 additional sequences are identified by allowing for the G:U wobble immediately 3′ to the deamination.

Figure 3.

MiR-513 and miR-769-3p target MAID sites but not the corresponding unedited sequence. (A) A diagram shows hairpin expression vectors and MAID reporter constructs. pAL-513 and pAL-769-3p reporter vectors have miR-513 and miR-769-3p hairpins downstream of the miR-517 Pol-III promoter (28). TAAT, TGAT and TGGT reporters contain three tandem copies of the 13 bp MAID sequence in the 3′-UTR of Renilla luciferase for testing activity in the unedited (TAAT) or edited (TGAT and TGGT) states. Guanines mimicking A-to-I edits are bolded and underscored. (B) Renilla luciferase activity (normalized to firefly luciferase and presented as percent mock transfected control) following co-transfection of miR-513, miR-769-3p, pooled miR-513 and miR-769-3p inhibitors and/or control miRNA inhibitor with the indicated reporters into HEK 293 cells (n = 3) is illustrated. *P < 0.005.

Figure 4.

Endogenous MAIDs are targets for miR-513 and miR-769-3p repression. (A) A cartoon depicts the DFFA 3′-UTR and the localization of nine distinct MAIDs (lines above the 3′-UTR). (B) Alignment of the nine DFFA 3′-UTR MAIDs commonly deaminated in ESTs is represented. MAID sequences are shaded. Four MAIDs contain G:U wobbles from consensus (bold). (C) Alignment of DFFA_1 sequences from three independent DFFA clones isolated from HEK 293 cells and NB7 cells is shown. DFFA_1 was deaminated in NB7s (bold) but not in HEK 293 cells. RT reactions were performed using a thermostable reverse transcriptase. (D) A diagram of DFFA 3′-UTR reporter constructs is shown. In DFFA-Edited (-E) and DFFA-Unedited (-U), the Renilla luciferase 3′-UTRs are the cloned DFFA 3′-UTRs from NB7 and HEK 293 cells, respectively. DFFA-E nucleotides differing from DFFA-U are bolded and underscored (compare NB7_2 and 293_1 detailed in panel (C). (E) Luciferase assays performed identically to those in Fig. 3B except for the reporter constructs illustrated (n = 3). *P < 0.005.

Figure 5.

MiR-769-3p selectively represses DFFA protein. (A) Relative mature miR-769-3p levels in HEK 293, A549, HT1080 and NB7 cell lines are shown as determined by quantitative PCR. MiR-513 was not detected in these cell lines. (B) Western blot analysis of endogenous DFFA in HEK 293 and NB7 cell lysates following transfection of miR-769-3p as indicated. Representative blots for DFFA and β-catenin (loading control) are shown. (C) Relative DFFA levels calculated as band intensity ratios of DFFA to β-catenin and normalized to mock (left most bar in each graph). 400, 200 and 100 refer to ng of miR-769-3p expression vector.