Functional characterization of mammalian microRNAs

描述（由申请人提供）：最近随着微RNA（miRNAs）的发现，出现了基因表达调控的新范例，微RNA是进化上保守的一类小的（约22个核苷酸-nt-）调控RNA。miRNA与其mRNA靶点部分或广泛互补结合，miRNA通过抑制翻译或通过内切核酸裂解使其mRNA靶点不稳定来控制基因表达，miRNA可以在基因表达调控中发挥深远的作用，因为它们具有靶向许多mRNA的能力，miRNA在功能上等同于小干扰RNA（siRNA）。使用合成的siRNA来敲低基因表达已经改变了基础生物学研究，并且如果解决了关于siRNA的递送和特异性的问题，则有望彻底改变医学实践。 尽管最近mi/siRNA领域的爆炸性增长，但关于哺乳动物miRNAs功能的许多重要问题仍然没有答案，并形成了该提议的基础。在目标1中，我们将鉴定和表征mi/siRNA引导的核糖核酸内切酶（RNAi核糖核酸内切酶）。我们提出的部分亲和纯化和这种酶的性质和生化方法，将使我们能够识别和表征这种重要的酶。我们还提出了研究结果和策略，以阐明mi/siRNA如何识别其mRNA靶标。在目标2中，我们将分析miRNA指导的翻译抑制的因素和机制。我们已经开始鉴定与miRNA相关的蛋白质，当它们识别多聚核糖体中的同源mRNA靶标时，我们已经产生了一个体外系统，该系统重现了哺乳动物miRNA指导的翻译抑制。我们目前的方法来表征哺乳动物的miRNA介导的翻译抑制。我们希望我们的研究将促进我们对RNAi的分子机制和miRNA在人类中的功能的理解。

DESCRIPTION (provided by applicant): A new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs), an evolutionary conserved class of small (approximately 22 nucleotide -nt-), regulatory RNAs. miRNAs bind with partial or extensive complementarity to their mRNA targets, miRNAs control gene expression by repressing the translation or by destabilizing, by endonucleolytic cleavage, their mRNA targets, miRNAs may exert profound effects in gene expression regulation as they have the capacity to target numerous mRNAs, miRNAs are functionally equivalent to small interfering RNAs (siRNAs). The use of synthetic siRNAs to knockdown gene expression has already transformed basic biology research and is promising to revolutionize the practice of medicine, if issues regarding delivery and specificity of siRNAs are solved. Despite the recent, explosive growth in the mi/siRNA field, many important questions regarding the function of mammalian miRNAs remain unanswered and form the basis of this proposal. In Aim 1, we will identify and characterize the mi/siRNA-guided endoribonuclease (RNAi endoribonuclease). We present the partial affinity purification and properties of this enzyme and biochemical approaches that will allow us to identify and characterize this important enzyme. We also present results and strategies to elucidate how mi/siRNAs recognize their mRNA targets. In Aim 2, we will analyze the factors and mechanisms underlying miRNA-directed translational repression. We have begun the identification of proteins that miRNAs associate with, when they recognize their cognate mRNA targets in polyribosomes, and we have generated an in vitro system that recapitulates mammalian miRNA-directed translational repression. We present approaches to characterize translational repression mediated by mammalian miRNAs. We expect that our studies will promote our understanding of the molecular mechanisms underlying RNAi and the function of miRNAs in humans.