Defining the Boundaries of RNAi in a cell

定义细胞内 RNAi 的边界

• 批准号: 10396103
• 负责人: Krystal Courtney Johnson
• 金额: $ 3.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-11 至 2023-05-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396103
• 关键词: Applications Grants; Attention; Binding; Binding Sites; Biogenesis; Biological Models; Biological Process; Biology; Cell Line; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colon Carcinoma; Conflict (Psychology); Development; Disease; Dissection; Environment; Eukaryota; Experimental Designs; Fellowship; Fostering; Gene Expression; Gene Expression Regulation; Gene Silencing; Genome; Goals; Guidelines; HCT116 Cells; Hypoxia; Infrastructure; Knock-out; Laboratories; Literature; Location; Malignant Neoplasms; Mammalian Cell; Manuscripts; Mass Spectrum Analysis; Measures; Mediating; Mentorship; Messenger RNA; MicroRNAs; Modeling; Outcome; Oxidative Stress; Paper; Pathway interactions; Physiology; Proteins; PubMed; Publications; Publishing; RNA; RNA Binding; RNA Interference; Regulation; Reporting; Repression; Research; Resources; Role; Scientist; Signal Pathway; Site; Stress; Techniques; Testing; Training; Untranslated RNA; Work; Writing; base; cell growth regulation; cell type; experimental study; human disease; mRNA Expression; miRNA expression profiling; multidisciplinary; nervous system disorder; nutrient deprivation; overexpression; overexpression analysis; paralogous gene; protein expression; response; therapeutic miRNA; tool; transcriptome sequencing; tumor

Project Summary RNA interference (RNAi) has driven a surge of research over the past twenty years as a powerful tool for gene silencing, and the discovery of a distinct class of noncoding RNAs, microRNAs (miRNAs), has revolutionized our outlook of genome regulation. miRNAs bind to a critical effector molecule, the Argonaute protein, to achieve sequence-specific recognition and repression of their target. Because the rules for complementary recognition seem simple and predictable, the dysregulation of miRNAs has been implicated as the sole culprit of disease in over 40,000 articles on PubMed. The sheer volume of publications suggest that miRNAs have omnipotent control over gene expression. This model of ubiquitous miRNA regulation suggested that there is an urgent need for a fundamental reexamination of the available gene silencing infrastructure present in a cell. Progress in identifying high-quality candidates for miRNA-based therapies have been hindered by this major barrier that miRNAs are expected to recognize more targets in a cell than there are catalytic molecules to guide. This project seeks to answer the question of how the competition for shared gene silencing resources can have a dramatic impact on the number of targets miRNAs can control. The first and second aim will both use quantitative mass spectrometry (MS) to count the AGO molecules per cell and RNA-sequencing to determine the miRNA and mRNA expression in a HCT116 colon cancer model system. While the first aim will interrogate CRISPR-mediated AGO knockout cell lines, the second aim will provide the complementary overexpression analysis. Furthermore, these cell lines will be used as rigorous controls for unbiased identification of the AGOs’ respective RNA binding sites using AGO-eCLIP-Seq. The third aim is to use three types of cell stress, nutrient deprivation, oxidative stress, and hypoxia, to trigger an endogenous RNAi response, which will be assessed by AGO copy number with quantitative MS, miRNA and target expression with RNA-Seq techniques, and RNA binding locations with AGO-eCLIP-Seq. The multi-disciplinary environment in the Corey laboratory fosters collaboration amongst diverse backgrounds, and trainees benefit from Dr. Corey’s attentive mentorship and the guidance of several established research scientists. By the end of this training fellowship period, I will accomplish four goals: 1) to be knowledgeable about the literature in my chosen field of RNA biology, 2) to be comfortable writing grant proposals and manuscripts, 3) to be technically competent, and 4) speak with clarity to all audiences.

项目摘要 RNA干扰（RNAi）作为一种强大的技术，在过去二十年中推动了研究的激增 基因沉默的工具，以及一类独特的非编码RNA，微小RNA（miRNA）， 彻底改变了我们对基因组调控的看法miRNAs与一个关键的效应分子结合， Argonaute蛋白，实现序列特异性识别和阻遏其靶点。因为 互补识别的规则似乎简单而可预测， 在PubMed上的4万多篇文章中，它被认为是疾病的唯一罪魁祸首。数量庞大 的出版物表明，miRNA对基因表达具有全能的控制。这个型号的 普遍存在的miRNA调控表明，迫切需要进行根本性的重新检查， 基因沉默基础结构的最大可能性。 鉴定基于miRNA的治疗的高质量候选物的进展受到阻碍 通过这一主要障碍，预期miRNA在细胞中识别的靶点比 催化分子来引导。该项目旨在回答如何竞争的问题， 共享的基因沉默资源可以对靶miRNA的数量产生巨大影响， 控制第一个和第二个目标都将使用定量质谱（MS）来计数AGO 分子/细胞和RNA测序，以确定HCT 116中的miRNA和mRNA表达。 结肠癌模型系统。虽然第一个目标将询问CRISPR介导的AGO敲除细胞， 线，第二个目标将提供互补的过表达分析。此外，这些细胞 细胞系将用作严格对照，用于无偏鉴定AGO各自的RNA结合 使用AGO-eCLIP-Seq.第三个目标是使用三种类型的细胞压力，营养剥夺， 氧化应激和缺氧，以触发内源性RNAi反应，这将通过以下方法进行评估： AGO拷贝数与定量MS，miRNA和靶表达与RNA-Seq技术，和 使用AGO-eCLIP-Seq的RNA结合位置 科里实验室的多学科环境促进了不同领域之间的合作。 背景，学员受益于博士科里的细心指导和指导的几个 建立科学研究。在这段培训期结束时，我将实现四个目标： 1)了解我所选择的RNA生物学领域的文献，2）舒适 撰写拨款申请和手稿，3）技术能力，4）清晰地向所有人说话 受众