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RNA Editing of Human Micrornas Open Access Research2006BlowetVolume al. 7, Issue 4, Article R27 RNA editing of human microRNAs comment Matthew J Blow*, Russell J Grocock†, Stijn van Dongen†, Anton J Enright†, Ed Dicks*, P Andrew Futreal*, Richard Wooster* and Michael R Stratton*‡ Addresses: *Cancer Genome Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. †Computational and Functional Genomics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. ‡Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, SM2 5NG, UK. Correspondence: Michael R Stratton. Email: [email protected] reviews Published: 4 April 2006 Received: 8 December 2005 Revised: 30 January 2006 Genome Biology 2006, 7:R27 (doi:10.1186/gb-2006-7-4-r27) Accepted: 6 March 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/4/R27 © 2006 Blow et al.; licensee BioMed Central Ltd. reports This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human<p>Atargets.</p> survey miRNA of RNAediting editing of miRNAs from ten human tissues indicates that RNA editing increases the diversity of miRNAs and their Abstract deposited research Background: MicroRNAs (miRNAs) are short RNAs of around 22 nucleotides that regulate gene expression. The primary transcripts of miRNAs contain double-stranded RNA and are therefore potential substrates for adenosine to inosine (A-to-I) RNA editing. Results: We have conducted a survey of RNA editing of miRNAs from ten human tissues by sequence comparison of PCR products derived from matched genomic DNA and total cDNA from the same individual. Six out of 99 (6%) miRNA transcripts from which data were obtained were research refereed subject to A-to-I editing in at least one tissue. Four out of seven edited adenosines were in the mature miRNA and were predicted to change the target sites in 3' untranslated regions. For a further six miRNAs, we identified A-to-I editing of transcripts derived from the opposite strand of the genome to the annotated miRNA. These miRNAs may have been annotated to the wrong genomic strand. Conclusion: Our results indicate that RNA editing increases the diversity of miRNAs and their targets, and hence may modulate miRNA function. interactions Background development [10]. It is estimated that up to 30% of human MicroRNAs (miRNAs) are short (around 20-22 nucleotides) genes may be miRNA targets [11,12]. RNAs that post-transcriptionally regulate gene expression by base-pairing with complementary sequences in the 3' miRNAs are transcribed by RNA polymerase II into long pri- untranslated regions (UTRs) of protein-coding transcripts mary miRNA (pri-miRNA) transcripts which are capped and and directing translational repression or transcript degrada- polyadenylated [13,14]. Genomic analyses indicate that many information tion [1-5]. There are currently 326 human miRNAs listed in miRNAs overlap known protein coding genes or non-coding the miRNA registry version 7.1 [6], but the total number of RNAs [15], and that many are in evolutionarily conserved miRNAs encoded in the human genome may be nearer 1,000 clusters with other miRNAs [16]. Furthermore, intronic miR- [7,8]. The function of most miRNAs is unknown, but many NAs share expression patterns with adjacent miRNAs and the are clearly involved in regulating differentiation [9] and host gene mRNA indicating that they are coordinately coex- pressed [17]. Genome Biology 2006, 7:R27 R27.2 Genome Biology 2006, Volume 7, Issue 4, Article R27 Blow et al. http://genomebiology.com/2006/7/4/R27 Pri-miRNAs contain a short double-stranded RNA (dsRNA) sequences were next oriented with respect to the strand of stem-loop formed between the miRNA sequence and its adja- transcription of the miRNAs. In six cases the A-to-G changes cent complementary sequence. In the nucleus, the ribonucle- were in the same orientation as the miRNA, and overlap the ase III-like enzyme Drosha cleaves at the base of this stem- stem-loop structure of the miRNA, consistent with RNA edit- loop to liberate a miRNA precursor (pre-miRNA) as a 60-70- ing of the pri-miRNA precursor transcript. In an additional nucleotide RNA hairpin [18]. The pre-miRNA hairpin is case, A-to-I editing was observed in a novel stem-loop struc- exported to the cytoplasm by exportin-5 [19-21] where it is ture in sequence adjacent to the unedited miRNA miR-374. further processed into a short dsRNA molecule by a second This novel stem-loop structure may represent a novel miRNA ribonuclease III-like enzyme, Dicer [22-24]. A single strand (Figure 2, novel hairpin). In the remaining five cases, the A- of this short dsRNA, the mature miRNA, is incorporated into to-G changes were from the opposite strand to the miRNA a ribonucleoprotein complex. This complex directs transcript (that is, U-to-C changes in the miRNA sequence). Although cleavage or translational repression depending on the degree U-to-C editing of miRNA sequences cannot be ruled out, no of complementarity between the miRNA and its target site. editing of this type has previously been observed and no enzymes capable of catalyzing this conversion are known. The RNA editing is the site-specific modification of an RNA most likely explanation is that these are A-to-I edits in a tran- sequence to yield a product differing from that encoded by the script derived from the DNA strand complementary to the DNA template. Most RNA editing in human cells is adenosine annotated miRNA gene. Consistent with this hypothesis, all to inosine (A-to-I) RNA editing which involves the conversion of these sequences overlap, or are adjacent to, genes tran- of A-to-I in dsRNA [25,26]. A-to-I RNA editing is catalyzed by scribed from the opposite strand to the annotated miRNA the adenosine deaminases acting on RNA (ADARs). The gene. To distinguish these sequences from the edited pri- majority of A-to-I RNA-editing sites are in dsRNA structures miRNAs, these sequences are referred to here as edited anti- formed between inverted repeat sequences in intronic or sense pri-miRNAs (Table 1). One of the antisense pri-miRNAs intergenic RNAs [25,27-30]. Therefore, the double-stranded contains editing sites overlapping the intended miRNA (miR- precursors of miRNAs may be substrates for A-to-I editing. 144) and miR-451, a recently identified miRNA that was not Indeed, it has recently been shown that the pri-miRNA tran- deliberately included in our list of 231 miRNAs. script of human miRNA miR-22 is subject to A-to-I RNA edit- ing in a number of human and mouse tissues [31]. Although Collectively the 13 sequences were edited at 18 sites. Ten out the extent of A-to-I editing was low (less than 5% across all of the 13 were edited at a single site. miR-376a and antisense adenosines analyzed), targeted adenosines were at positions miR-451 were each edited at two sites, and antisense miR-371 predicted to influence the biogenesis and function of miR-22. was edited at four sites. The extent of editing varied with edit- This raises the possibility that RNA editing may be generally ing site and with tissue, ranging from around 10% (for exam- important in miRNA gene function [31]. In this study we have ple, miR-151 in multiple tissues) to around 70% (antisense systematically investigated the presence of RNA editing in miR-371 in placenta). Overall, the levels of RNA editing miRNAs. observed were considerably higher than the approximately 5% editing previously reported for the -1 position of miR-22 [31]. Editing of miR-22 was not detectable by our method, Results presumably because the low levels of editing of this miRNA To search for RNA-editing sites in human miRNAs, PCR fall below our limits of detection. All miRNAs were found to product sequencing was performed from matched total cDNA be edited in multiple tissues, with the extent of editing vary- and genomic DNA isolated from adult human brain, heart, ing from tissue to tissue (Figure 1). liver, lung, ovary, placenta, skeletal muscle, small intestine, spleen and testis. Primers were designed to amplify pri- All novel A-to-I editing sites were found within the dsRNA miRNA sequences flanking all 231 human miRNAs in miR- stems of the predicted stem-loop structures (Figure 2). Of the Base [6]. Of these, 99 miRNA containing sequences were suc- seven editing sites in pri-miRNAs, four were in the 22-nucle- cessfully sequenced in both directions and from duplicate otide mature miRNA. Three of these were within nucleotides PCR products from total cDNA of at least one tissue. Total 2 to 7, which are thought to be important for conferring bind- cDNA sequence traces were compared with genomic DNA ing-site specificity between the miRNA and its target sites [3]. sequence traces from the same individual, and A-to-I editing Five out of seven editing sites in pri-miRNAs were at single was identified as an A in the genomic DNA sequence com- nucleotide A:C mismatches flanked by paired bases. Simi- pared with a novel G peak at the equivalent position in the larly, five out of seven editing sites were in 5'-UAG-3' trinucle- total cDNA sequence. otides. These results are consistent with local structural and sequence preferences of RNA editing determined from A-to-I In total, 12 of the 99 miRNA-containing sequences (13%) editing sites in inverted repeat sequences [25].
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