MicroRNAs (miRNAs) are endogenous ~23-nt RNAs that can play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.

We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets.

MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2-7), particularly those in 3Ј untranslated regions (3ЈUTRs), are preferentially conserved. Here, we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3ЈUTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites. The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the "offset 6mer," to be detected. In total, >45,000 miRNA target sites within human 3ЈUTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3Ј end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (P CT ) correlating with experimental measurements of repression. Our expanded set of target predictions (including conserved 3Ј-compensatory sites), are available at the TargetScan website, which displays the P CT for each site and each predicted target.[Supplemental material is available online at www.genome.org.]MicroRNAs are ∼22-nucleotide (nt) endogenous RNAs that derive from distinctive hairpin precursors in plants and animals (Bartel 2004). After incorporation into a silencing complex, which contains at its core an Argonaute protein, an miRNA can pair to an mRNA and thereby specify the post-transcriptional repression of that protein-coding message, either by transcript destabilization, translational repression, or both. MicroRNAs constitute one of the more abundant classes of gene-regulatory molecules in animals, with hundreds of distinct miRNAs confidently identified in both human and mouse (Landgraf et al. 2007). A central goal for understanding the functions of all these small regulatory RNAs has been to determine which messages are targeted for repression.The search for biological targets of metazoan miRNAs has benefited greatly from the comparative analysis of orthologous mRNAs. Targets of miRNAs can be predicted above the background of false-positive predictions by requiring conserved Watson-Crick pairing to the 5Ј region of the miRNA, known as the miRNA seed (Lewis et al. 2003). Because so many messages have preferentially preserved their pairing to miRNA seeds, targets can be predicted simply by searching for c...

MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks.DOI: http://dx.doi.org/10.7554/eLife.05005.001

Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are approximately 7 nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13-16, positioning within the 3'UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets.

MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.