Development of technologies for genome-wide identification of RNA branch points

描述(申请人提供)：表达20,000多个人类基因需要平均每个信使核糖核酸剪接8-10个内含子，大多数人类基因通过选择性剪接产生多种不同的信使核糖核酸和蛋白质亚型。基因组中200,000多个内含子中的每一个都包含3个特定的序列位点--供体或5‘剪接点、受体或3’剪接点和分支点--这是绝对必需的，因为它们参与了剪接的化学过程。分支点是一种特定的核苷酸(通常是腺苷)，它参与剪接的第一个催化步骤，产生独特的“套索内含子结构，该结构在剪接的第二步中释放。分支位点的突变经常导致外显子跳跃、内含子保留或正常剪接的其他干扰，这可能导致截短或异常蛋白的产生，有时还会导致疾病。然而，只有几十个人类内含子的分支点被绘制出来。在这里，我们建议开发一种大规模绘制RNA分支点的技术，使用模式生物来测试和优化该方法，然后应用优化后的程序在人和小鼠的基因组范围内绘制分支点。我们的建议围绕以下具体目标组织： SA1.制定大规模识别分支点的协议和相关的绘图软件，并适用于模式生物(酵母、苍蝇或蠕虫)。 SA2.优化并将SA1的协议和软件应用于哺乳动物系统，以实现对人类和小鼠基因组中分支点的大规模识别。 我们设计了两个分子生物学方案，当与第二代测序和相关的软件管道相结合时，有可能在全基因组范围内识别分支点。这项技术的发展和对蠕虫、苍蝇、人类和小鼠基因组的应用，有可能在我们理解这些生物的RNA剪接密码方面贡献一个关键的“缺失片段”，并将能够改进对通过干扰分支点功能而扰乱剪接和基因表达的突变或其他遗传变异的预测。 与公共卫生相关：该项目寻求开发一种技术，用于绘制RNA分支点的全基因组图谱，这些分支点是几乎每一个人类基因正确表达所必需的基因组特征。对分支点的大规模绘制将有助于更深入地了解基因表达所涉及的机制，并将能够更好地预测通过扰乱RNA分支点的功能而导致人类疾病的突变和其他遗传变异。

DESCRIPTION (provided by applicant): Expression of the full complement of 20,000+ human genes requires splicing of an average of 8-10 introns per mRNA, and most human genes produce multiple distinct mRNA and protein isoforms through alternative splicing. Each of the ~200,000+ introns in the genome contains 3 specific sequence sites - the donor or 5' splice site, the acceptor or 3' splice site and the branch point - that are absolutely required because they participate in the chemistry of splicing. The branch point is a specific nucleotide (usually adenosine) that participates in the first catalytic step of splicing, generating the unique "lariat intron structure that is released in the second step of splicing. Mutation of the branch site frequently results in exon skipping, intron retention or other perturbation of normal splicing, which can result in production of truncated or aberrant proteins, and sometimes leads to disease. However, branch points have been mapped for only several dozen human introns. Here, we propose to develop a technology to map RNA branch points on a large scale, using model organisms to test and optimize the method, followed by application of the optimized procedure to map branch points genome-wide in human and mouse. Our proposal is organized around the following specific aims: SA1. Develop a protocol for large-scale identification of branch points and associated mapping software and apply to model organisms (yeast, fly, or worm). SA2. Optimize and apply protocols and software from SA1 to mammalian systems to achieve large- scale identification of branch points in the human and mouse genomes. We have designed two molecular biology protocols that when coupled with second-generation sequencing and associated software pipelines have the potential to identify branch points on a genome-wide scale. Development of this technology and application to the worm, fly, human and mouse genomes has the potential to contribute a critical "missing piece" in our understanding of RNA splice codes in these organisms, and will enable improved prediction of mutations or other genetic variations that perturb splicing and gene expression by interfering with branch point function. PUBLIC HEALTH RELEVANCE: This project seeks to develop a technology for genome-wide mapping of RNA branch points, which are genomic features that are required for the proper expression of nearly every human gene. Large-scale mapping of branch points will lead to deeper understanding of the mechanisms involved in gene expression, and will enable improved predictions of mutations and other genetic variations that contribute to human disease by disrupting the function of RNA branch points.