Gene-centered protein-RNA interaction mapping

描述(申请人提供)：基因表达调控对健康发育和生理至关重要，许多疾病是由基因表达变化引起的或与之相关。在过去的十年中，已经收集了大量关于转录因子转录控制的信息，这些转录因子直接与其靶基因内或周围的调控DNA序列结合。此外，在转录后控制基因表达的microRNAs已经被广泛研究。然而，关于绝大多数RNA结合蛋白的功能和生化信息一直缺乏，尽管有明确的证据表明它们在发育和疾病中的重要性。导致我们对RNA结合蛋白转录后调控了解有限的一个主要因素是缺乏适当的技术，这些技术从一个重要的mRNA开始，例如对应于一个感兴趣的疾病基因，并识别与该mRNA相互作用的RNA结合蛋白。在拟议的项目中，我们将开发一种新的、高通量的方法来检测和识别RNA-蛋白质相互作用。我们暂时将这项技术命名为“RNA相关蛋白相互作用检测”(RAPID)。RAPID以翻译为基础，模拟内源RNA结合蛋白的活性。我们将首先开发和应用线虫中快速的RNA-蛋白质相互作用，因为它为进一步的体内研究提供了一个非常合适的模型，而且因为我们有可用的克隆资源，如ORFeome，它包含许多全长RNA结合蛋白编码克隆。该项目的成功完成将为研究界提供一种新的、广泛适用的方法，以无偏见和高通量的方式检测RNA-蛋白质相互作用。我们设想将快速应用于基因组规模的这种相互作用的检测，以加深我们对复杂基因调控网络的理解。我们将为线虫开发的方法学和RNA结合蛋白资源将为在其他模式生物和人类中创建类似资源提供重要的蓝图。

DESCRIPTION (provided by applicant): The regulation of gene expression is vital for healthy development and physiology, and many diseases are caused by or associated with changes in gene expression. In the past decade, a tremendous amount of information has been gathered regarding transcriptional control by transcription factors that bind directly to regulatory DNA sequences in or around their target genes. In addition, microRNAs that control gene expression post-transcriptionally have been studied extensively. However, functional and biochemical information about the vast majority of RNA binding proteins has been lacking despite clear evidence of their importance in development and disease. A major factor contributing to our limited understanding of post- transcriptional control by RNA binding proteins is a shortage of appropriate technologies that start with an important mRNA, for instance corresponding to a disease gene of interest, and identify the RNA binding proteins with which this mRNA interacts. In the proposed project, we will develop a novel, high-throughput method for the detection and identification of RNA-protein interactions. We have provisionally named this technology "RNA-associated protein interaction detection" (RAPID). RAPID is based on translation and mimics endogenous RNA binding protein activity. We will first develop and apply RAPID to RNA-protein interactions in the nematode Caenorhabditis elegans because it provides a highly suitable model for further in vivo studies, and because we have clone resources such as the ORFeome available, which contains numerous full-length RNA binding protein-encoding clones. Successful completion of this project will provide the research community with a novel and broadly applicable method to detect RNA-protein interactions in an unbiased and high-throughput manner. We envision applying RAPID to the genome-scale detection of such interactions to further our understanding of complex gene regulatory networks. The methodology and RNA binding protein resource that we will develop for C. elegans will provide an important blueprint for the creation of similar resources in other model organisms and humans.