Developmental regulation by miRNAs

描述(申请人提供)：microRNAs(MiRNAs)最近被描述为一类重要的新调节分子，控制无脊椎动物分化和模式的各个方面。在脊椎动物中也发现了类似的miRNAs，但它们在高等生物中的功能在很大程度上仍不清楚。这项建议旨在了解miRNAs在脊椎动物发育中的作用。我们发现，某些miRNAs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中的结构中表达的所有miRNAs，包括在发育中的肢芽中表达的miRNAs，包括在前肢和后肢原基中差异表达的miRNAs，以及在心脏原基中不对称产生的那些。这些miRNAs的空间分布将通过Northern印迹和携带“传感器”结构的瞬时转基因小鼠的产生来确定，这种“传感器”结构被设计成在除特定miRNA存在的地方以外的任何地方表达LacZ。将制造更多的传感器来检查单个miRNA家族(Iet7)的所有类似物的差异表达模式，以及基因组中编码在一个簇中的几个不同的miRNAs的差异表达模式。基于这些表达模式，将利用逆转录病毒错误表达在雏鸡中解决miRNAs的功能。最后，我们将研究1个miRNAs家族miR196在调节小鼠HOX基因功能中的作用。3个miR196基因座将被单独敲除，构建三个突变体。这些MIR调节HoxB8 mRNA的稳定性，并调节HoxA7 mRNA的翻译。HoxB8 3‘非编码区中的miR196靶序列将被敲入法删除，以确定这一调控的发育意义，并且HoxA7 3’非编码区中的miR196靶序列将被HoxB8 miR198靶序列取代，以测试调控模式(RNA切割与翻译控制)是否在功能上起作用。

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.