MOTIVATION: The massive spread of repetitive elements in the human genome presents a substantial challenge to the organism, as such elements may accidentally contain seemingly functional motifs. A striking example is offered by the roughly one million copies of Alu repeats in the genome, of which ∼0.5% reside within genes' untranslated regions (UTRs), presenting ∼30 000 novel potential targets for highly conserved microRNAs (miRNAs). Here, we examine the functionality of miRNA targets within Alu elements in 3'UTRs in the human genome. RESULTS: Using a comprehensive dataset of miRNA overexpression assays, we show that mRNAs with miRNA targets within Alus are significantly less responsive to the miRNA effects compared with mRNAs that have the same targets outside Alus. Using Ago2-binding mRNA profiling, we confirm that the miRNA machinery avoids miRNA targets within Alus, as opposed to the highly efficient binding of targets outside Alus. We propose three features that prevent potential miRNA sites within Alus from being recognized by the miRNA machinery: (i) Alu repeats that contain miRNA targets and genuine functional miRNA targets appear to reside in distinct mutually exclusive territories within 3'UTRs; (ii) Alus have tight secondary structure that may limit access to the miRNA machinery; and (iii) A-to-I editing of Alu-derived mRNA sequences may divert miRNA targets. The combination of these features is proposed to allow toleration of Alu insertions into mRNAs. Nonetheless, a subset of miRNA targets within Alus appears not to possess any of the aforementioned features, and thus may represent cases where Alu insertion in the genome has introduced novel functional miRNA targets. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of "target hubs" genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR-TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR-TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional-post transcriptional regulatory network.