Functional characterization of mammalian microRNAs

描述（由申请人提供）：随着microRNAs （miRNAs）的发现，最近出现了一种新的基因表达调控范式，microRNAs是一种进化保守的小分子（约22个核苷酸-nt-）调控rna。miRNAs与mRNA靶点部分或广泛互补结合，miRNAs通过抑制mRNA靶点的翻译或破坏稳定，通过核内溶裂解控制基因表达，miRNAs具有靶向多种mRNA的能力，可能在基因表达调控中发挥深远的作用，miRNAs在功能上相当于小干扰rna （sirna）。使用合成的sirna来抑制基因表达已经改变了基础生物学研究，如果sirna的传递和特异性问题得到解决，将有望彻底改变医学实践。

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.