A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency.
Bottom Line: Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome.The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency.These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.
Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.Show MeSH
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Mentions: To analyze miRNA-induced global changes in gene expression, mRNAs were divided into four groups based on the presence in their 3′-UTRs of at least one sequence complementary to the seed region containing either A, U, G or C at the possible editing site, respectively. The mRNAs that belonged to more than one of these groups, and those with 3′-UTR complementarity to the dead seed were eliminated to select for targets that may be downregulated by only one type of seed sequence. These separate groups are subsequently referred to as (I) U-target, (II) A-target, (III) C-target, (IV) G-target mRNAs (see Supplementary Figures S1–S3). Consequently, A-type miRNAs are expected to specifically downregulate ‘U-target’ genes, whereas the I-type and G-type miRNAs should downregulate ‘C-target’ genes. The background constitutes of all mRNAs—excluding dead seed targets—that are outside of each individual group. As a result, the background differs slightly between the four groups. The procedure for microarray data analysis is illustrated in Figure 2A. At first, we made an MA plots showing the mean log2 of signal intensities relative to those of mock transfection (M value) and the averaged log10 signal intensities of mock and miRNA transfections (A value), and plotted these values as the vertical and horizontal bars, respectively (Figure 2A(1)). To facilitate understanding, the MA plot was then converted to the cumulative distribution (Figure 2A(2)), in which the horizontal axis indicates the ‘M value’ and the vertical axis is the cumulative fraction of mRNAs. The mean log2 of signal intensities relative to those of mock transfection was calculated for each group of mRNAs and the results are shown as MA plots and cumulative distributions (Supplementary Figures S1–S3). Furthermore, to simplify the results, the ‘area’ between seed-matched mRNAs (red curve in Figure 2A(2)) and background RNAs (black curve in Figure 2A(2)) was calculated and shown as the ‘fold-change’ in Figure 2B–G, with positive values meaning that the target genes are generally more inhibited than background. Furthermore, the Wilcoxon rank-sum test was used to assess whether or not the target genes and background distributions are significantly different (p < 1×10−2).
Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.