Didiano Dominic, Hobert Oliver
Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University Medical Center, New York, New York 10032, USA.
RNA. 2008 Jul;14(7):1297-317. doi: 10.1261/rna.1082708. Epub 2008 May 7.
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.
动物基因组包含数百种微小RNA（miRNA），即小型调节性RNA，它们通过与靶mRNA中的互补位点结合来控制基因表达。一些支配miRNA/靶标相互作用的规则已得到阐明，但其普遍适用性仍有待根据具体情况进行进一步实验验证。我们在此使用转基因线虫中的一种检测系统来分析秀丽隐杆线虫lsy-6 miRNA与3' UTR序列的相互作用。与许多先前用于分析miRNA/靶标相互作用的检测系统不同，我们的检测系统在lsy-6正常发挥功能的细胞环境中运行，该环境是秀丽隐杆线虫神经系统中的单个神经元。通过广泛的突变分析，我们确定了lsy-6已知且经实验验证的靶标（cog-1同源框基因的3' UTR）中对于功能性miRNA/靶标相互作用所必需的特征。我们描述了无论是在cog-1 3' UTR的背景下还是在异源3' UTR的背景下，一个或多个种子匹配并非功能性miRNA/靶标相互作用的可靠预测指标。我们反而发现，miRNA靶标位点之外的两个非序列特异性的上下文特征是miRNA介导的3' UTR调控的关键决定因素。这些上下文特征位于3' UTR中lsy-6结合位点的下游，并以组合方式起作用；每个特征的突变导致3' UTR调控的缺陷有限，但组合缺失则导致3' UTR调控完全丧失。与两个lsy-6位点一起，这两个上下文特征能够赋予异源3' UTR调控能力。此外，上下文特征需要相对于miRNA结合位点以特定构型存在，并且可能代表蛋白质结合位点或提供合适的结构背景。我们得出结论，给定的靶标位点存在于3' UTR背景中，该背景的进化超越了靶标位点互补性，以支持特定miRNA的调控。我们在本研究中分析的大量3' UTR对于计算生物学家设计下一代miRNA/靶标预测算法也将是有用的。
