Transcriptome-wide Identification and Validation of Interactions between the miRNA Machinery and HuR on mRNA Targets

The 3′ untranslated region (UTR) of mRNAs is the primary regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins in the cytoplasm. Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3′ UTR sites have emerged. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RNA-binding protein, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3′ UTRs and assess their co-occurrence. In addition, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2–HuR overlap, 13 candidates were screened in luciferase reporter assays. Eleven sites showed miRNA-dependent repression, as confirmed in Dicer-null cells. To test for HuR's role in co-regulation, we measured the reporters in HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA–HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3′ UTR. [Display omitted] •Ago2 occupancy on mRNAs with and without HuR, and vice versa, is measured by CLIP-seq.•Ago2 interacts with HuR at the level of binding.•In addition to antagonism, cooperative interactions are observed transcriptome-wide.•Ago2–HuR overlap loci are prevalent and possess characteristics of genuine sites.•Individual validation demonstrates cooperative and antagonistic regulation.