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. 2010 Sep 7;107(36):15751-6.
doi: 10.1073/pnas.1006172107. Epub 2010 Aug 20.

Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs

Michael Schnall-Levin  1 , Yong Zhao, Norbert Perrimon, Bonnie Berger
Affiliations

Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs

Michael Schnall-Levin et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate protein-coding genes posttranscriptionally. In animals, most known miRNA targeting occurs within the 3'UTR of mRNAs, but the extent of biologically relevant targeting in the ORF or 5'UTR of mRNAs remains unknown. Here, we develop an algorithm (MinoTar-miRNA ORF Targets) to identify conserved regulatory motifs within protein-coding regions and use it to estimate the number of preferentially conserved miRNA-target sites in ORFs. We show that, in Drosophila, preferentially conserved miRNA targeting in ORFs is as widespread as it is in 3'UTRs and that, while far less abundant, conserved targets in Drosophila 5'UTRs number in the hundreds. Using our algorithm, we predicted a set of high-confidence ORF targets and selected seven miRNA-target pairs from among these for experimental validation. We observed down-regulation by the miRNA in five out of seven cases, indicating our approach can recover functional sites with high confidence. Additionally, we observed additive targeting by multiple sites within a single ORF. Altogether, our results demonstrate that the scale of biologically important miRNA targeting in ORFs is extensive and that computational tools such as ours can aid in the identification of such targets. Further evidence suggests that our results extend to mammals, but that the extent of ORF and 5'UTR targeting relative to 3'UTR targeting may be greater in Drosophila.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MicroRNA seed sites are among the most highly conserved motifs in coding regions. (A) Histogram of conservation scores for all 65,536 8-mers. Nearly all of the top-conserved 8-mers correspond to miRNA seed sites. (B) Cumulative plot of scores for different sets of 8-mers. Shown are all 8-mers (black), conserved Drosophila miRNA seeds (red), reverse complements of these seeds (green), 8-mers with identical dinucleotide content to these seeds (cyan), nonconserved Drosophila miRNA seeds (blue), and conserved human miRNA seeds (magenta). (C) Imposing increasingly stringent conservation cutoff results in higher signal-to-background ratios for the set of Drosophila conserved miRNA seeds, whereas control sets behave as background at all cutoffs. (D) Conservation of seed sites accompanied by 3′ base-pairing to the miRNA starting at different positions within the miRNA, as compared to the background conservation of seed sites.
Fig. 2.
Fig. 2.
The scale of conserved miRNA targeting in 3′UTRs, ORFs, and 5′UTRs. (A) Fraction of sites conserved above background for both 8-mers and 7-mers in 3′UTRs, ORFs, and 5′UTRs. (B) Number of predicted sites above background for 8-mers and 7-mers in 3′UTRs, ORFs, and 5′UTRs. Error bars show standard deviation in the estimates obtained from sampling of background sets (see Methods).
Fig. 3.
Fig. 3.
MicroRNA seeds showing the highest level of conservation in 3′UTRs tend also to be the most conserved in ORFs and in 5′UTRs, but not in promoter regions. Shown are the fractions of 8-mer sites conserved above background at 60% confidence cutoff (for 5′UTRs and promoters the cutoff was chosen to be the same as for 3′UTRs) among (A) 3′UTRs and ORFs, (B) 3′UTRs and 5′UTRs, and (C) 3′UTRs and promoters. Dotted vertical and horizontal lines show the cutoff for conservation above background equal to the maximal amount by which any miRNA was conserved below background.
Fig. 4.
Fig. 4.
Experimental verification of target predictions. (A) Illustration of the experiment. Each ORF with wild-type miRNA-target site and the same ORF with mutated site were placed under different epitope tags and coexpressed with either a control plasmid or miRNA, and then run on Western blot. Quantification of down-regulation was made by comparing the ratio of the two channels under miRNA vs. under control plasmid. In all cases, epitope tags were flipped to confirm the effect was consistent under each set of tags. Shown are the bands from a test of CG11178 targeting by mir-1. (B) Down-regulation of target genes. Shown are the fold changes of targets under targeting miRNAs (red bars) as well as under miRNAs not predicted to target the genes (blue bars). Error bars show standard deviation, asterisks denote p values (Student’s t test; **: p < 0.01, ***: p < 0.001). (C) Effect of multiple target sites. Shown is down-regulation of CG8494 by mir-1 with all eight combinations of the three predicted sites (WT sites are marked as + and mutated sites as −), averaged over three separate experiments. Error bars give standard deviation.
Fig. 5.
Fig. 5.
Predicted ORF sites are preferentially down-regulated. Shown is the cumulative distribution for genes with no sites (black), genes with a conserved ORF site (red), genes with any ORF site (blue), genes with a conserved 3′UTR site (magenta), and genes with any 3′UTR site (cyan). Genes with conserved ORF sites show down-regulation about twice as strong as those with any ORF site, and about half as strong as those with predicted 3′UTR sites.
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