Functional RNA elements in the human genome

描述(申请人提供)：该项目旨在系统地解码人类基因组中的功能RNA元件。该方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。Fu博士和Yeo博士在RNA研究方面拥有广泛的实验和计算专业知识，他们将共同领导团队实施拟议的三个具体目标：(1)我们建议将CLIP(交联式免疫沉淀)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置，最初专注于在构成和调控的前mRNA加工中涉及的RNA结合蛋白。这一结果有望对人类受调控的RNA加工产生新的生物学和机械学见解。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，系统地产生针对人类基因组中编码的RNA结合蛋白的抗体。(2)在物理作图的同时，我们将把我们实验室以前开发的寡核苷酸选择/连接策略与高通量测序结合起来，进行选择性剪接的数字分析。在经历了所有基于阵列的检测mRNA异构体的系统后，我们相信，目前的实验设计将通过克服与基于阵列的方法相关的所有技术问题，产生关于调控剪接的高灵敏度和高特异性的定量信息。(3)我们将对目标1和目标2中产生的物理图谱和功能图谱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大规模的基因组序列分析相结合，例如通过计算预测的RNA元件、基因组间的保守性以及与已知剪接位点的耦合。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总体理解做出独特的贡献。 项目简介：人类基因的表达在DNA和RNA水平上都受到调控。作为对大多数致力于破译功能DNA元件的努力的补充，这一建议侧重于通过对一大组RNA结合蛋白的物理定位以及对替代的mRNA异构体表达的功能分析来研究人类基因组中的功能RNA元件。实验产生的数据将与其他大规模分析计算推断的RNA元件相结合，以了解RNA水平调控基因表达背后的一般原理，这很有可能揭示人类细胞调控剪接的密码，并为各种疾病机制提供新的线索。

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.