Double-Stranded RNA and Antisense Regulation in Vivo

描述（由申请人提供）：本提案的总体目标是开发表征体内互补RNA分子之间稳定碱基配对相互作用的方法。这些方法将用于表征哺乳动物细胞内双链RNA的存在，以分析反义调控的机制。尽管反义调控已经在几个原核生物系统中得到了充分的记录和机制探索，但关于反义rna改变真核生物中互补转录物表达的机制，我们所知的相对较少。然而，越来越多的证据表明，哺乳动物基因组的许多区域表达重叠，这些重叠来自染色体的两条链。在某些情况下，这种转录可能引起反义调控，其中目标转录物的表达通过与互补反义RNA的碱基配对而受到抑制。本研究有两个具体目标：(1)建立在体内形成的双链RNA的分离、鉴定和表征方法；(2)从分离的dsRNA中制备扩增的cdna文库，这些文库将使用随机或靶向策略进行筛选。该策略的一个关键特征是，单链rna将在分离之前进行修饰，以防止不确定退火。为了方便鉴定分离天然双链RNA的最佳条件，一个合成的RNA发夹将在转染的胚胎癌细胞中表达，作为特异性Aim 1的靶序列。最后，将使用基因特异性探针和高通量方法筛选文库中的互补重叠序列，以表征稳定的双链rna群体。这些研究结果将为深入了解RNA-RNA碱基配对相互作用在体内的形成和稳定性，以及进一步研究这种相互作用的功能作用所需的方法和信息提供依据。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop methods for characterizing stable base-pairing interactions between complementary RNA molecules in vivo. These methods will be used to characterize the presence of double-stranded RNA within in mammalian cells in order to analyze mechanisms for antisense regulation. Although antisense regulation has been well-documented and mechanistically explored in several prokaryotic systems relatively little is known regarding the mechanism by which antisense RNAs alter the expression of complementary transcripts in eukaryotes. However, there is accumulating evidence that many regions of the mammalian genome expressed overlapping that are transcribed from both strands of the chromosome. In some instances this transcription may give rise to antisense regulation in which expression of a target transcript is repressed by base pairing with complementary antisense RNA present. The proposed study has two specific goals: (1) to develop methods for the isolation, identification and characterization of doublestranded RNA formed in vivo; and (2) to prepare libraries of amplified cDNAs from isolated dsRNA that will be screened using either random or targeted strategies. A key feature of this strategy is that single-stranded RNAs will be modified prior to isolation to prevent adventitious annealing. To facilitate identification of condition that are optimal for isolation of native double-stranded RNA, a synthetic RNA hairpin will express in transfected embryonic carcinoma cells to serve as a target sequence in Specific Aim 1. Finally, libraries will be screened for complementary overlapping sequences using both gene specific probes and high throughput methods to characterize the population of stable double-stranded RNAs. The results of these studies will provide insight into the formation and stability of RNA-RNA base-paired interactions in vivo, and the methods and information needed for further studies of the functional roles of such interactions.