Variant induced RNA structure change in human genetic disease

人类遗传病中变异诱导的RNA结构变化

• 批准号: 10410412
• 负责人: Alain T Laederach
• 金额: $ 41.64万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10410412
• 关键词: 3-Dimensional; Acylation; Alleles; Biological; Biological Assay; Cells; Chemical Structure; Chemicals; Data; Disease; Disease model; Elements; Etiology; Gene Expression; Gene Expression Regulation; Genetic Diseases; Genetic Transcription; Goals; Human Genetics; Hybrids; Hydroxyl Radical; Maps; Measures; Messenger RNA; Modeling; Modification; Molecular; Mutation; Organism; Primer Extension; Process; Proteins; RNA; RNA Splicing; RNA Stability; RNA-Directed DNA Polymerase; Reagent; Reporter; Research; Role; Sampling; Structure; Sum; Technology; Translations; Untranslated RNA; Variant; Viral Genome; Virus; Work; crosslink; experimental study; genetic variant; genome-wide; human disease; human tissue; in vivo; innovation; novel; programs; technological innovation; transcriptome

SUMMARY My research program focuses on quantitatively characterizing the role of RNA structure in post-transcriptional regulatory processes. Comparisons of protein and messenger RNA (mRNA) abundance at genome scale reveal low correlation between the two gene expression levels in most human tissues and other organisms. This poor correlation suggests that a significant amount of gene regulation occurs post-transcriptionally. To discover elements in mRNAs that control their activities, we measure the effects of human disease-associated structure variants that map to non-coding regions of the transcriptome. Specifically, we integrate computational structure prediction with high-throughput allele-specific chemical structure probing in vivo to assess the functional consequences of RNA structure change. We also establish the causality of these variants by using quantitative reporter assays to measure translation efficiency, splicing, and mRNA stability. In total, these experiments provide molecular explanations of disease mechanisms. To support these goals, we develop, implement, and apply both computational and experimental approaches to study RNA structure in the cell. Our proposed research program will further develop two important technological innovations. The first is a hybrid experimental/computational approach for studying precursor and mature mRNA structure simultaneously in vivo; this approach integrates mutational profiling of SHAPE-MaP data (Selective 2’-Hydroxyl Acylation by Primer Extension – Mutational Profiling) with Boltzmann suboptimal sampling of the secondary structural ensemble. The second innovation is SHAPE-JuMP, which uses a bifunctional RNA modification reagent and a highly processive reverse transcriptase that “jumps” across chemical crosslinks to probe through-space three-dimensional contacts in RNA. We will use these technologies to establish the structures of both precursor and mature mRNAs. In addition, we will extend the biological scope of our work by using these technologies to collaboratively investigate inter- and intramolecular interactions in positive strand RNA viruses. In sum, this program will identify novel RNA structure motifs that regulate the functions of precursor and mature mRNAs and viral genomes.

概括 我的研究计划的重点是定量表征RNA结构在转录后的作用 监管过程。蛋白质和信使RNA（mRNA）在基因组量表上的抽象比较揭示了 大多数人体组织和其他生物的两个基因表达水平之间的低相关性。这个穷人 相关性表明，转录后发生了大量基因调节。发现 控制其活动的mRNA中的元素，我们测量与人类疾病相关结构的影响 映射到转录组的非编码区域的变体。具体来说，我们整合了计算结构 用高通量等位基因特异性化学结构在体内进行的预测以评估功能 RNA结构的后果变化。我们还通过使用定量来建立这些变体的因果关系 记者测定法测量翻译效率，剪接和mRNA稳定性。总共这些实验 提供疾病机制的分子解释。为了支持这些目标，我们开发，实施和 应用计算方法和实验方法研究细胞中的RNA结构。我们提出的 研究计划将进一步发展两项重要的技术创新。第一个是混合动力 用于研究前体和成熟的mRNA结构的实验/计算方法很容易在体内； 这种方法整合了形状映射数据的突变分析（通过底漆选择性2'-羟基酰基化 扩展 - 突变分析），辅助结构合奏的玻尔兹曼次优采样。这 第二个创新是Shape-Jump，它使用双功能的RNA修饰试剂和高度的处理方法 逆转录酶在化学交联中“跳跃”以探测通过空间三维触点探测 在RNA中。我们将使用这些技术来建立前体和成熟mRNA的结构。在 此外，我们将通过使用这些技术协作调查来扩展工作的生物学范围 阳性链RNA病毒中分子间和分子内相互作用。总而言之，该程序将识别新型RNA 调节前体和成熟mRNA和病毒基因组功能的结构基序。