Developmental regulation by miRNAs

描述（由申请人提供）：MicroRNAs （miRNAs）最近被描述为一类重要的新调控分子，控制着无脊椎动物的分化和模式。在脊椎动物中也发现了类似的mirna，但它们在高等生物中的功能在很大程度上仍然未知。这一建议旨在了解mirna在脊椎动物发育过程中的作用。我们发现某些mirna在肢体和心脏的形态发生过程中存在差异表达。我们将使用微阵列和mini-Sage方法来鉴定在这些发育结构中表达的所有mirna，包括鉴定在发育中的肢体芽中表达的mirna，包括在前肢和后肢原基中差异表达的mirna以及在心脏原基中左右不对称产生的mirna。这些miRNA的空间分布将通过Northern blot和瞬时转基因小鼠的生产来确定，这些小鼠携带“传感器”结构，设计用于在除给定miRNA存在的地方外的任何地方表达lacZ。将制作额外的传感器来检查单个miRNA家族的所有旁系的差异表达模式（Iet7）；以及基因组中一个簇中编码的几种不同的mirna。基于这些表达模式，mirna的功能将通过逆转录病毒错表达在小鸡中得到解决。最后，我们将研究1个mirna家族miR196在小鼠Hox基因功能调控中的作用。将3个miR196位点分别敲除，构建三重突变体。这些miRs调节HoxB8 mRNA的稳定性并调节HoxA7 mRNA的翻译。HoxB8 3'UTR中的miR196靶序列将通过敲入法被删除，以确定该调控的发育意义；HoxA7 3'UTR中的miR196靶序列将被HoxB8 miR198靶序列所取代，以测试调控模式（RNA切割与翻译控制）是否在功能上有影响。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have recently been described as an important new class of regulatory molecules controlling aspects of differentiation and patterning in invertebrates. Similar miRNAs have been discovered in vertebrates, but their function in higher organisms remains largely unknown. This proposal is directed towards understanding the role of miRNAs during vertebrate development. We have found that certain miRNAs are differentially expressed during morphogenesis of the limb and heart. We will use microarray and mini-Sage approaches to identify all miRNAs expressed in these developing structures, including the identification of miRNAs expressed in the developing limb buds, including those differentially expressed in the fore- and hind-limb primordia as well as those left-right asymetrically produced in the heart primordium. The spatial distribution of these miRNAs will be determined by Northern blot and by production of transient transgenic mice carrying "sensor" constructs which are designed to express lacZ everywhere except where a given miRNA is present. Additional sensors will be made to examine the differential expression patterns of all paralogues of a single miRNA family (Iet7); and also of several distinct miRNAs encoded in a cluster in the genome. Based on these expression patterns, the function of miRNAs will be addressed in the chick using retroviral misexpression. Finally, we will study the roles of 1 family of miRNAs, miR196, in regulating Hox gene function in mice. The 3 miR196 loci will be knocked-out individually and the triple mutant will be constructed. These miRs regulate stability of the HoxB8 mRNA and modulate translation of the HoxA7 mRNA. The miR196 target sequence in the HoxB8 3'UTR will be deleted by a knock-in approach to determine the developmental significance of this regulation and the miR196 target sequence in the HoxA7 3'UTR will be replaced with the HoxB8 miR198 target sequence to test whether the mode of regulation (RNA cleavage versus translational control) functionally matters.