Gene-centered protein-RNA interaction mapping

描述（由申请人提供）：基因表达的调节对健康发育和生理至关重要，许多疾病是由基因表达的变化引起的或与之相关。在过去的十年中，已经收集了大量的信息，关于直接结合到其靶基因中或周围的调节DNA序列的转录因子的转录控制。此外，转录后控制基因表达的microRNA已被广泛研究。然而，关于绝大多数RNA结合蛋白的功能和生物化学信息一直缺乏，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结合蛋白的转录后控制的理解有限的主要因素是缺乏适当的技术，该技术从重要的mRNA开始，例如对应于感兴趣的疾病基因，并鉴定与该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鉴定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基于翻译并模拟内源性RNA结合蛋白活性。我们将首先开发和应用RAPID的线虫的RNA-蛋白质相互作用，因为它提供了一个非常合适的模型，进一步在体内研究，因为我们有克隆资源，如ORFeome，其中包含许多全长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颖且广泛适用的方法，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RAPID应用于这种相互作用的基因组规模检测，以进一步了解复杂的基因调控网络。本研究将为C.秀丽线虫将为在其他模式生物和人类中创造类似资源提供重要的蓝图。 公共卫生相关性：基因表达的调节对于健康发育和稳态是至关重要的，并且许多疾病是由基因表达的变化引起的或与基因表达的变化相关。近年来，在研究单个RNA结合蛋白及其如何影响基因表达方面取得了巨大进展。然而，基因组编码数百种这样的蛋白质，并且缺乏能够同时表征许多蛋白质的方法。拟议的项目是开发一种新技术，该技术将广泛适用于RNA结合蛋白的功能和大规模表征，并将影响基因表达的系统生物学领域和研究一个或几个疾病相关人类基因的研究人员。

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. PUBLIC HEALTH RELEVANCE: The regulation of gene expression is vital for healthy development and homeostasis and many diseases are caused by or associated with changes in gene expression. In recent years, tremendous progress has been made in the study of individual RNA binding proteins and how they affect gene expression. However, the genome encodes hundreds of such proteins, and methods that enable the characterization of many at one time are lacking. The proposed project is to develop a novel technology that will be broadly applicable for the functional and large-scale characterization of RNA binding proteins, and will impact both the fields of systems biology of gene expression and researchers that study one or a few disease-relevant human genes.