Defining the Boundaries of RNAi in a cell

项目摘要 在过去的二十年里，RNA干扰(RNAi)作为一种强大的 基因沉默的工具，以及发现一类不同的非编码RNA，microRNAs(MiRNAs)， 彻底改变了我们对基因组调控的看法。MiRNAs结合到一个关键的效应分子，即 ArgAerte蛋白，实现序列特异性识别和抑制其靶标。因为 互补性识别的规则似乎简单而可预测，即miRNAs的失调 在PubMed上的40,000多篇文章中被认为是疾病的唯一罪魁祸首。巨大的体量 的出版物表明，miRNAs对基因表达具有全能的控制。这一模式 普遍存在的miRNA调控表明，迫切需要进行根本性的重新审查。 细胞中存在的可用的基因沉默基础设施。 在确定基于miRNA的疗法的高质量候选者方面的进展受到了阻碍 通过这一主要障碍，miRNAs有望识别细胞中比细胞内更多的靶标 催化分子来引导。这个项目试图回答这样一个问题，即竞争如何 共享的基因沉默资源可以对miRNAs可以达到的靶点数量产生巨大影响 控制力。第一个和第二个目标都将使用定量质谱学(MS)来计算过去 每个细胞的分子数和RNA测序以确定HCT116中miRNA和mRNA的表达 结肠癌模型系统。虽然第一个目标是询问CRISPR介导的AGO基因敲除细胞 线，第二个目的将提供补充过度表达分析。此外，这些细胞 LINE将被用作严格的控制，以无偏见地识别AGOS各自的RNA结合 使用AGO-eCLIP-Seq.的站点第三个目标是使用三种类型的细胞应激，营养剥夺， 氧化应激和缺氧，以触发内源性RNAi反应，这将通过以下方式进行评估 用定量MS、miRNA和RNA-Seq技术进行靶表达 RNA与AGO-eCLIP-Seq的结合位置。 科瑞实验室的多学科环境促进了不同学科之间的合作 背景，受训者受益于科里博士的用心指导和几个 老牌的研究科学家。到本次培训团契结束时，我将实现四个目标： 1)了解我选择的RNA生物学领域的文献，2)熟悉 撰写赠款提案和手稿，3)具备技术能力，4)清晰地表达所有人的意思 观众。

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.