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 在过去的二十年中，RNA干扰（RNAI）作为一个强大的 基因沉默的工具，发现了不同编码的RNA，microRNA（miRNA），发现的工具 已经彻底改变了我们对基因组调节的看法。 miRNA与关键效应分子结合， Argonaute蛋白，以实现其靶标的序列特异性识别和表达。因为 完全识别的规则似乎简单且可预测，miRNA的失调 在PubMed上有40,000篇文章中，隐含是疾病的唯一罪魁祸首。纯粹的音量 出版物表明，miRNA对基因表达具有万能控制。这个模型 无处不在的miRNA法规表明，迫切需要进行基本重新审查 细胞中存在的可用基因沉默基础设施。 识别基于miRNA的疗法的高质量候选者的进展已受到阻碍 通过这一主要障碍，预计miRNA将识别细胞中更多的靶标的 催化分子引导。该项目旨在回答有关如何竞争的问题 共享的基因沉默资源可能会对miRNA可以的目标数量产生巨大影响 控制。第一个和第二个目标都将使用定量质谱（MS）来计数AGO 每个细胞的分子和RNA顺序确定HCT116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介导的以前的淘汰单元 线，第二个目标将提供互补的过表达分析。此外，这些细胞 线将用作严格的控件，以无偏鉴定AGO的各自的RNA结合 使用AGO-ECLIP-SEQ的站点。第三个目的是使用三种类型的细胞应力，营养剥夺， 氧化应激和缺氧引发内源性RNAi反应，将通过 具有定量MS，miRNA和目标表达的AGO拷贝数，带有RNA-seq技术，并且 RNA结合位置具有AGO-ECLIP-SEQ。 科里实验室的多学科环境促进了潜水员的合作 背景和受训者受益于科里博士的细心心态和几个的指导 建立的研究科学家。到这一培训奖学金期结束时，我将完成四个目标： 1）要了解我选择的RNA生物学领域的文献，2）舒适 编写赠款建议和手稿，3）在技术上有能力，4）对所有人说话 受众。