RNA editing of a miRNA precursor

Abstract

Micro RNAs comprise a large family of small, functional RNAs with important roles in the regulation of protein coding genes in animals and plants. Here we show that human and mouse miRNA22 precursor molecules are subject to posttranscriptional modification by A-to-I RNA editing in vivo. The observed editing events are predicted to have significant implications for the biogenesis and function of miRNA22 and might point toward a more general role for RNA editing in the regulation of miRNA gene expression.

FIGURE 1.

A-to-I RNA editing of miRNA22. (A) The exon/intron structure of the human miRNA22 gene as deduced from expressed sequences with GenBank accession numbers. Exons are shown as boxes, introns as lines. The location of the mature miRNA22 sequence and the positions of the oligonucleotide primers used for editing analysis of two different splice variants A and B are indicated. (B) Predicted RNA secondary structure of pre-miRNA22 with the mature miRNA sequence underlined. Edited positions in human and/or mouse tissues (filled arrowheads) and additional sites found only in ADAR1 overexpressing cell lines (open arrowheads) are indicated. (C) Editing levels of miRNA22 precursor molecules in various human tissues, human cell lines, wild-type mouse brain, and brains of ADAR2 knock-out mice. A PCR product derived from the human miRNA22 genomic region (gDNA), as well as the reverse transcribed and amplified coding sequences from human CUTL1 (CCAAT displacement proteinlike) and DISK1 (disrupted in Schizophrenia 1) genes, were analyzed as negative controls. The number of individually sequenced clones is indicated above each column. In addition to the total extent of editing (percent of molecules edited at one or more of the 17 adenosines within the pre-miRNA), the results for the +1 position of miRNA22 are shown because they point toward a pronounced site selectivity of ADAR2 versus ADAR1. For RNA editing analysis, total RNA was isolated from human brain (Harvard Brain Tissue Collection) using Trizol Reagent (Invitrogen) and reverse transcribed with Superscript Reverse Transcriptase II (Invitrogen) and N₆ random oligonucleotides as described (Maas et al. 2001). Genomic DNA was isolated from the same specimen. Additional sources for cDNA preparation were mouse brain total RNA prepared from SV129 mice, human lung and testis RNAs (Clontech), and total RNA extracted from stably transfected human cell lines (Maas et al. 2001). cDNA from ADAR2 knock-out mice (Higuchi et al. 2000) were a kind gift from Miyoko Higuchi and Peter H. Seeburg (Heidelberg). Primers miRNA22D (5′-TGAGGAGCCTGTTCCTCTCACG-3′) and miRNA22U (5′-CGCACTATGGTGCCACATCTCG-3′) for cDNA and primers gmiRNA22D (5′-GGAATTCCTTAGGAGCCTGTTCCTCTCACG-3′) and gmiRNA22U (5′-CCAGGAATCTAGAGTCTGGGC-3′) for genomic DNA were used for miRNA22-specific PCRs. Amplicons were subcloned into pBSII vector plasmid (Stratagene) and recombinant clones were isolated by blue–white selection. At least 100 individual clones were sequenced (C-tracked) from each subcloned PCR fragment. Splice form A was analyzed for all samples and splice variant B where detectable. The theoretical background of A/G discrepancies resulting from errors during reverse transcription and PCR was calculated as P = a · c + b · c · d = 3.4 · 10⁻⁵ · 17 + 0.8 · 10⁻⁵ · 17 · 24 = 0.4 · 10⁻², with a being the error rate of Superscript reverse transcriptase (3.4 · 10⁻⁵; Potter et al. 2003), b the error rate of Taq DNA polymerase (0.8 · 10⁻⁵; Cline et al. 1996), c the number of adenosines analyzed, and d the number of template doublings during PCR (estimated for 30 cycles of amplification). We would therefore expect 0.4 base changes affecting adenosine in 100 sequences, corresponding to 1 base change due to mutation in 250 sequences. Because changes of A to C and A to T are also possible, this error rate is likely an overestimation because the analysis only detects A-to-G changes. The results from analysis of genomic DNA isolated from human brain is in line with this value, as within 200 sequences clones we did not detect a single A/G change. Analysis of genomic DNA from HEK293 cells yielded the same result (data not shown), as well as RT-PCR and sequencing of coding regions from human CUTL1 (BC025422) and DISC1 (AK025293) RNAs.