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

RNA分支点全基因组鉴定技术的开发

• 批准号: 8310598
• 负责人: CHRISTOPHER B BURGE
• 金额: $ 26.81万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-24 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310598
• 关键词: 3' Splice Site; 5' Splice Site; Adenosine; Alternative Splicing; Animal Model; Biological; Biological Assay; Caenorhabditis elegans; Chemistry; Code; Complement; Complementary DNA; Computer software; Coupled; Data; Data Set; Development; Disease; Drosophila melanogaster; Enzymes; Eukaryota; Expressed Sequence Tags; Gel; Gene Expression; Generations; Genes; Genetic Variation; Genome; Genomics; Human; Human Cell Line; Human Genome; Introns; Lead; Libraries; Location; Mammalian Cell; Maps; Messenger RNA; Methods; Modeling; Molecular Biology; Mus; Mutation; Myoblasts; Nematoda; Nucleotides; Organism; Other Genetics; Procedures; Process; Production; Property; Protein Isoforms; Proteins; Protocols documentation; RNA; RNA Interference; RNA Splicing; Reading; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Ribosomes; Role; Saccharomyces cerevisiae; Sensitivity and Specificity; Site; Small RNA; Spliced Genes; Staging; Structure; System; Technology; Testing; Transcript; Yeasts; base; cancer cell; cell type; design; exon skipping; fly; genome wide association study; genome-wide; human disease; improved; interest; markov model; mouse genome; ribonuclease R; technology development

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.

描述（由申请人提供）： 20,000 多个人类基因的完整表达需要每个 mRNA 平均 8-10 个内含子的剪接，并且大多数人类基因通过选择性剪接产生多种不同的 mRNA 和蛋白质亚型。基因组中约 200,000 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' 剪接位点、受体或 3' 剪接位点以及分支点 - 这些是绝对必需的，因为它们参与剪接化学。分支点是特定的核苷酸（通常是腺苷），参与剪接的第一步催化，产生独特的“套索内含子结构”，并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扰动，这可能导致截短或异常蛋白质的产生，有时会导致疾病。然而，分支点仅被映射为 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，使用模型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分支点。我们的提案围绕以下具体目标进行组织： SA1。开发大规模识别分支点和相关绘图软件的协议，并将其应用于模型生物（酵母、苍蝇或蠕虫）。 SA2。将SA1的协议和软件优化并应用到哺乳动物系统中，以实现人类和小鼠基因组中分支点的大规模识别。 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使用时，有可能在全基因组范围内识别分支点。这项技术的开发和应用于蠕虫、苍蝇、人类和小鼠基因组有可能为我们理解这些生物体中的RNA剪接代码贡献一个关键的“缺失的部分”，并且将能够改进对通过干扰分支点功能来扰乱剪接和基因表达的突变或其他遗传变异的预测。 公共健康相关性：该项目旨在开发一种用于 RNA 分支点全基因组图谱的技术，这些分支点是几乎每个人类基因正确表达所需的基因组特征。分支点的大规模绘图将有助于更深入地了解基因表达所涉及的机制，并将能够改进对通过破坏 RNA 分支点的功能而导致人类疾病的突变和其他遗传变异的预测。