A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence

Abstract

Recognition sites for microRNAs (miRNAs) have been reported to be located in the 3 untranslated regions of transcripts. In a computational screen for highly conserved motifs within coding regions, we found an excess of sequences conserved at the nucleotide level within coding regions in the human genome, the highest scoring of which are enriched for miRNA target sequences. To validate our results, we experimentally demonstrated that the let-7 miRNA directly targets the miRNA-processing enzyme Dicer within its coding sequence, thus establishing a mechanism for a miRNA/Dicer autoregulatory negative feedback loop. We also found computational evidence to suggest that miRNA target sites in coding regions and 3 UTRs may differ in mechanism. This work demonstrates that miRNAs can directly target transcripts within their coding region in animals, and it suggests that a complete search for the regulatory targets of miRNAs should be expanded to include genes with recognition sites within their coding regions. As more genomes are sequenced, the methodological approach that we used for identifying motifs with high sequence conservation will be increasingly valuable for detecting functional sequence motifs within coding regions.computational biology ͉ posttranscriptional regulation ͉ comparative genomics ͉ multiple-sequence alignment ͉ evolutionary conservation M icroRNAs (miRNAs) are endogenously encoded, singlestranded regulatory RNAs that bind to and inhibit the translation of transcripts with complementary sequence (1). Computational evidence suggests that miRNAs regulate at least 20% of human genes and have been implicated in the regulation of a wide range of biological systems (2). In plants, miRNA targets can be predicted with relatively high confidence because of the extensive base pairing between plant miRNAs and their target mRNAs (1). In animals, in contrast, miRNAs typically bind to their targets with significantly less complementarity, and the short target sequences are, therefore, difficult to identify on the basis of sequence alone. As a result, most computational approaches to predict miRNA-target interactions rely on conservation of target sites (3-6).Although early studies reported some evidence for the targeting of miRNAs to sites within protein coding regions (4, 6), subsequent research has reported that there is minimal functionality for sites in ORFs or 5Ј UTRs (7). A focus on miRNAs present within 3Ј UTRs is supported by evidence suggesting that the G-cap/poly(A) tail interface (which connects the two ends of eukaryotic mRNAs during translation) is important for miRNA function (8) and that miRNAs tend to be more effective when localized at the end of the 3Ј UTR rather than the middle (7, 9). Indeed, the protein translation machinery might be expected to displace an miRNA complex present within a gene's coding sequence. However, exogenously added siRNAs that target coding sequences, including siRNAs with imperfect base pairing, are effective at silencing (10). More recent reports have also shown that, c...

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