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

描述（由申请人提供）：20，000+人基因的完整补体的表达需要每个mRNA平均剪接8-10个内含子，并且大多数人基因通过选择性剪接产生多种不同的mRNA和蛋白质同种型。基因组中的~ 200，000+内含子中的每一个包含3个特定的序列位点-供体或5'剪接位点、受体或3'剪接位点和分支点-这是绝对需要的，因为它们参与剪接的化学过程。分支点是一个特定的核苷酸（通常是腺苷），它参与了剪接的第一个催化步骤，产生了独特的“内含子"结构，在剪接的第二个步骤中释放出来。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，这可导致截短或异常蛋白质的产生，并且有时导致疾病。然而，分支点已经被定位为只有几十个人类内含子。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，然后应用优化的程序来绘制人类和小鼠全基因组的分支点。我们的建议是围绕以下具体目标提出的： SA 1.开发一个大规模识别分支点的方案和相关的绘图软件，并应用于模式生物（酵母、苍蝇或蠕虫）。 SA 2.优化和应用从SA 1到哺乳动物系统的协议和软件，以实现人类和小鼠基因组中分支点的大规模鉴定。 我们设计了两种分子生物学方案，当与第二代测序和相关软件管道结合时，它们有可能在全基因组范围内识别分支点。这项技术的发展和应用于蠕虫，苍蝇，人类和小鼠基因组有可能在我们理解这些生物体中的RNA剪接密码中贡献一个关键的“缺失片段”，并将能够改善对突变或其他遗传变异的预测，这些突变或其他遗传变异通过干扰分支点功能来干扰剪接和基因表达。

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.