权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Defining the Boundaries of RNAi in a cell

定义细胞内 RNAi 的边界

基本信息

批准号：
10164564
负责人：
Krystal Courtney Johnson
金额：
$ 3万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/10164564
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

项目摘要

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.

项目总结