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多种人类基因的完整补体的表达需要每个mRNA平均8-10个内含子的剪接，并且大多数人基因通过替代剪接产生多个不同的mRNA和蛋白质同工型。基因组中的约200,000+内含子中的每个都包含3个特定序列位点 - 供体或5'剪接位点，受体或3'剪接位点和分支点 - 由于它们参与了剪接化学，因此绝对需要。分支点是参与剪接的第一个催化步骤的特定核苷酸（通常是腺苷），产生了在剪接第二步中释放的独特的“套管内含子结构”。分支部位的突变经常导致外显子跳过，内含子保留，内含子保留，固定或其他疾病的疾病，这些疾病的产生可能会导致造成截止的分支，有时会造成型号的蛋白质，并有时会遇到或异常的蛋白质，并且会导致疾病，并有时会遇到型号，并且有时会导致或依赖型物，并且会导致疾病，并有时会导致或近似型号，并且会导致疾病，并且会导致疾病，并且有时会导致型号，并且有时会导致疾病，并且有时会导致疾病，并且有时会导致疾病，并且会导致疾病的生产。几个人的内含子。我们建议开发一项技术，以大规模绘制RNA分支点，并使用模型生物来测试和优化该方法，然后将优化程序应用于人类和小鼠的范围内。 SA1。开发用于大规模识别分支点和相关映射软件的协议，并适用于模型生物（酵母，飞或蠕虫）。 SA2。优化并应用从SA1到哺乳动物系统的方案和软件，以实现人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结合时，有可能在全基因组范围内识别分支点。在我们对这些生物体中RNA剪接代码的理解时，这项技术和对蠕虫，苍蝇，人和小鼠基因组的应用有可能贡献一个关键的“缺失部分”，并可以通过干扰分支点功能来改善突变或其他遗传变异的预测，从而改善对突变或其他遗传变异的预测。 公共卫生相关性：该项目旨在开发一项用于RNA分支点的全基因组映射的技术，这是几乎每个人类基因的适当表达所需的基因组特征。分支点的大规模映射将导致对基因表达所涉及的机制的更深入的了解，并可以改善对突变和其他遗传变异的预测，这些变异通过破坏RNA分支点的功能，从而有助于人类疾病。