Abstract 11338: Analysis of the RISC Complex Reveals Canonical and Seedless Targets of miR-210 Hypoxamir
BACKGROUND: MicroRNA-210 (miR-210) is a key player of endothelial cell response to hypoxia, modulating glycolysis, VEGF-driven endothelial cell migration and the ability of endothelial cells to form capillary-like structures in vitro. Since miRNA function is mediated by the ability to bind and inhibit their target mRNAs, target identification is instrumental for understanding miRNA function.
METHODS AND RESULTS: To experimentally identify a large set of miR-210 targets, we used a biochemical approach based on the immunoprecipitation of RISC effector complexes (RISC-IP) enriched for miR-210 and consequently for its targets. miR-210 was expressed to levels similar to these observed in hypoxia and cellular extracts were immunoprecipitated for Ago2. Next, the co-immuno-precipitated mRNAs were measured by Illumina deep sequencing technology. Data analysis with a custom bioinformatic pipeline allowed the identification of 34 new targets. To validate these results, the enrichment of the new targets was confirmed by qPCR in miR-210 containing RISC-IPs. Next, we took the reciprocal approach and found that miR-210 targets were decreased in RISC-IPs derived from cells where miR-210 was inhibited by the transfection of a specific LNA-anti-miR. Moreover, 5 targets were further validated by luciferase reporter assays. Among the genes enriched in miR-210 RISC-IPs, we also identified ROD1. Surprisingly, ROD1 lacked the canonical complementarity with miR-210 seed sequence, an highly conserved region at miRNA 5’. However, a “centered” binding site in ROD1 involving 10 bases of miR-210 central portion was identified and validated by reporter assays. As expected, both ROD1 mRNA and protein were down-modulated upon hypoxia in a miR-210-dependent manner. Thus, ROD1 represents a bona fide miR-210 seedless target. Finally, ROD1 targeting by miR-210 was biologically significant: rescue of ROD1 down-modulation significantly increased >4 fold hypoxia-induced cell death. These data highlight the importance of ROD1 regulation by miR-210 for cell response to hypoxia.
CONCLUSIONS: The unbiased analysis of the mRNAs associated to miR-210-RISC allowed the identification of canonical and non canonical targets underpinning miR-210 biological function.
- © 2012 by American Heart Association, Inc.