Modulation of Ago-miRNA regulatory networks by cis-sequence elements and target competition

Abstract

regulators of gene expression in a wide range of organisms and biological processes. Each miRNA guides Argonaute (Ago) protein complexes to target and repress hundreds of genes in a sequence-dependent manner. To identify all targets of miRNA regulation, we performed UV crosslinking and immunoprecipitation (CLIP) of Ago complexes in mouse embryonic (ESC) and mesenchymal (MSC) stem cell lines. We also captured the genome-wide miRNA-independent binding footprint of Ago by performing CLIP in cells that lack Dicer, an enzyme required for mature miRNA biogenesis. We surprisingly found that Ago bound a similar set of genes in the absence of Dicer, and this overlap in target genes was due partially to residual, unprocessed miRNAs in the Dicer KO cells. Other potential sites of miRNA-independent Ago interactions, such as histone transcripts and poly-A cleavage and polyadenylation sites, were also identified. One Ago CLIP dataset revealed the enrichment for a G-rich sequence motif at Ago target sites. We later demonstrated that the G-motif is not directly bound to Ago but rather is enriched near miRNA-guided Ago binding sites. Its presence near miRNA target sites is associated with stronger repression of Ago-miRNA targets. Fortuitously, the original Ago CLIP dataset that identified the G-motif was later shown to likely contain target sites of another co-immunoprecipitating RNA binding protein (RBP). Using mass spectroscopy of Ago antibody immunoprecipitations from Ago KO cells, we identified a list of interacting RBPs that could potentially augment Ago-miRNA activity through the G-motif. To investigate target competition in miRNA networks, we related our CLIP analysis of genome-wide, quantitative Ago binding to measurements of absolute miRNA and target RNA concentrations. We found that all miRNAs other than the miR-290 family in ESCs and let-7 family in MSCs were expressed at concentrations below their total target pool. However, 8-12 miRNA families were expressed at near or greater than equimolar ratios with their pool of high affinity targets, and this affinity-partitioned stoichiometry led to significant Ago accumulation and repression of high affinity target sites despite little consequential binding at low affinity sites. Single-cell reporter assays demonstrated that high expressed miRNAs are not susceptible to physiological inductions of competing target transcripts but targets of lower expressed miRNAs are derepressed in a weakly threshold-like manner upon increased target pool levels. In summary, we identify a network of confidently bound targets of miRNA regulation in ESCs and MSCs, reveal the extent of miRNA-independent binding in these two cell types, provide a list of potential miRNA enhancer RBPs, and create a quantitative context for evaluating target competition in miRNA networks.