Functional RNA elements in the human genome

描述（申请人提供）：本项目旨在系统地解码人类基因组中的功能性RNA元件。该提案整合了几项最新的技术进步，并充分利用了我们实验室中Solexa系统的可用性。Drs。Fu和Yeo在RNA研究方面拥有广泛的实验和计算专业知识，并将共同领导团队开展该项目，具体目标有三个：(1)我们建议将CLIP（交联免疫沉淀）技术与高通量测序相结合，以确定大量RNA结合蛋白的体内结合位点，初步关注与构成型和受调节的pre-mRNA加工相关的RNA结合蛋白。该结果有望为人类受调节的RNA加工提供新的生物学和机制见解。为了准备该项目未来的扩展，我们正在与一家抗体生产公司合作，系统地生产针对人类基因组中编码的RNA结合蛋白的抗体。(2)在物理图谱绘制的同时，我们将把之前在实验室开发的寡核苷酸选择/连接策略与高通量测序结合起来，对选择性剪接进行数字分析。在经历了所有基于阵列的检测mRNA同种异构体的系统后，我们相信，通过克服与基于阵列的方法相关的所有技术问题，当前的实验设计将产生具有高灵敏度和特异性的调控剪接定量信息。(3)我们将对目标1和目标2中生成的物理图谱和功能分析数据进行生物信息学分析，以推断人类基因组中的调控RNA元件。这些数据将进一步与其他基因组序列的大规模分析相结合，例如计算预测的RNA元件，基因组间的保守性，以及与已知剪接位点的偶联。我们相信这个项目将对RNA基因组学以及我们对基因表达的转录后调控的理解做出独特的贡献。

DESCRIPTION (provided by applicant): This project is designed to systematically decode functional RNA elements in the human genome. The proposal integrates several recent technological advances and capitalizes the availability of the Solexa system in our lab. Drs. Fu and Yeo have extensive experimental and computational expertise in RNA research and will jointly lead the team to carry out the proposed project in three specific aims: (1) We propose to couple the CLIP (Crosslinking ImmunoPrecipitation) technology with high throughput sequencing to identify in vivo binding sites for a large number of RNA binding proteins, focusing initially on RNA binding proteins implicated in both constitutive and regulated pre-mRNA processing. The results are expected to generate novel biological and mechanistic insights into regulated RNA processing in humans. To prepare future expansion of the project, we are working with an antibody production company to systematically generate antibodies against RNA binding proteins encoded in the human genome. (2) In parallel to physical mapping, we will couple the oligo selection/ligation strategy previously developed in our lab with high throughput sequencing to perform digital analysis of alternative splicing. Having experienced all array-based systems for detecting mRNA isoforms, we believe that the current experimental design will generate quantitative information on regulated splicing with both high sensitivity and specificity by overcoming all technical problems associated with array-based approaches. (3) We will perform bioinformatics analysis of both physical mapping and functional profiling data generated in aim 1 and 2 to deduce regulatory RNA elements in the human genome. The data will be further integrated with other large-scale analysis of genomic sequences, such as computationally predicted RNA elements, conservation across genomes, and coupling with known splice sites. We believe that this project will make a unique contribution to RNA genomics as well as to our understanding of post-transcriptional regulation of gene expression in general. Project Narrative: The expression of human genes is regulated at both DNA and RNA levels. Complementary to most effort devoted to decipher functional DNA elements, this proposal focuses on functional RNA elements in the human genome by physical mapping of a large panel of RNA binding proteins coupled with functional analysis of alternative mRNA isoform expression. The experimentally generated data will be integrated with other large-scale analysis of computationally deduced RNA elements to understand general principle behind regulated gene expression at the RNA level, which has a high potential to reveal a code for regulated splicing in human cells and shed new light on various disease mechanisms.