Systematic analysis of the Hmga2 3′ UTR identifies many independent regulatory sequences and a novel interaction between distal sites

The 3' untranslated regions (3' UTRs) of mRNAs regulate transcripts by serving as binding sites for regulatory factors, including microRNAs and RNA binding proteins. Binding of such trans-acting factors can control the rates of mRNA translation, decay, and other aspects of mRNA biology. To better understand the role of 3' UTRs in gene regulation, we performed a detailed analysis of a model mammalian 3' UTR, that of Hmga2, with the principal goals of identifying the complete set of regulatory elements within a single 3' UTR, and determining the extent to which elements interact with and affect one another. Hmga2 is an oncogene whose overexpression in cancers often stems from mutations that remove 3'-UTR regulatory sequences. We used reporter assays in cultured cells to generate maps of cis-regulatory information across the Hmga2 3' UTR at different resolutions, ranging from 50 to 400 nt. We found many previously unidentified regulatory sites, a large number of which were up-regulating. Importantly, the overall location and impact of regulatory sites was conserved between different species (mouse, human, and chicken). By systematically comparing the regulatory impact of 3'-UTR segments of different sizes we were able to determine that the majority of regulatory sequences function independently; only a very small number of segments showed evidence of any interactions. However, we discovered a novel interaction whereby terminal 3'-UTR sequences induced internal up-regulating elements to convert to repressive elements. By fully characterizing one 3' UTR, we hope to better understand the principles of 3'-UTR-mediated gene regulation.