Molecular architecture of a miRNA-regulated 3' UTR.
Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.
Didiano
Dominic
D
Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, New York 10032, USA.
Hobert
Oliver
O
D020413:3' Untranslated Regions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001482:Base Composition; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018510:RNA, Helminth / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment
D020413:3' Untranslated Regions; D029742:Caenorhabditis elegans Proteins; D018398:Homeodomain Proteins; D035683:MicroRNAs; D018510:RNA, Helminth; C491329:cog-1 protein, C elegans