Recognition of Cellular Targets by single and double-stranded nucleic acids

摘要 背景：我的实验室工作在细胞生物学和核酸化学的接口， 调节翻译、剪接和转录。理想的项目有三个特点：1）新颖 寡核苷酸，使我们能够探测化学修饰的优点和缺点; 2） 用于核酸介导的调节的核酸结构或机制;以及 有机会深入了解重要的生物过程或治疗发展。2021年- 到2026年，我们的中心目标是研究RNA识别的机制，以更深入地了解 细胞生理学和治疗发展。 自2016年以来，已有多个核酸药物获批，寡核苷酸药物正在 准备对广泛的患者群体产生重大影响。实现这一点的一个障碍是 潜在的是对细胞中RNA识别的不完全理解。例如，当超过1000 由于每个月都有同行评议的出版物引用“miRNA”一词，我们缺乏详细的信息。 预测RNA功能或理解RNA在复杂细胞中的作用 生物过程。对拟议效果的验证往往是有问题的， 研究新基因是有限的。我们将集中在两个涉及RNA识别的项目领域 在人类细胞的细胞核或细胞质中。 RNAi过程中miRNAs与蛋白因子的协同作用我们将从根本上 使用缺乏关键RNAi因子的敲除细胞系深入了解RNA的机制，包括 Argonaute（AGO）和含三核苷酸重复的蛋白6（TNRC6）家族的蛋白。我们 将结合联合收割机质谱、RNAseq和交联免疫沉淀（CLIP）来定义 蛋白质或miRNAs的数量如何影响对基因调控的影响。 我们将首先研究细胞质中调控的详细机制。我们将应用这些 研究miRNA在细胞质和细胞核中识别的结果。 新机制和疾病靶点。我们将更好地了解两个 由突变RNA引起的疾病、富赫角膜内皮营养不良（FECD）和弗里德赖希角膜营养不良（Friedreich’s） 共济失调（FA）。目标之一是开发更有效的化合物以促进药物开发。第二 目的是更好地了解作用机制。这两种疾病都涉及核非编码RNA， 我们将使用合成核酸作为工具来探测疾病机制， 少量的突变RNA分子如何导致迟发性退行性疾病。

Abstract Background: My laboratory works at the interface of cell biology and nucleic acid chemistry to modulate translation, splicing, and transcription. Ideal projects have three features: 1) Novel oligonucleotides that allow us to probe the strengths and weakness of the chemical modifications; 2) Challenging nucleic acid structures or mechanisms for nucleic acid-mediated regulation; and 3) The opportunity to gain insights into significant biological process or therapeutic development. For 2021- 2026 our central goal is to investigate mechanisms of RNA recognition to gain greater insights into cell physiology and therapeutic development. Since 2016, several nucleic acid drugs have been approved and oligonucleotide medicine is poised to have a significant impact on broad patient populations. One obstacle to realizing this potential is an incomplete understanding of RNA recognition in cells. For example, while over 1000 peer-reviewed publications appear every month that cite the term “miRNA”, we lack the detailed insights necessary to necessary to predict function or appreciate the roles of RNA in complex biological processes. Validation of proposed effects is often problematic and the predictive power for studying novel genes is limited. We will focus on two project areas involving the recognition of RNA within either the nuclei or cytoplasm of human cells. Cooperation of miRNAs and protein factors during RNAi. We will gain fundamental insights into the mechanism of RNA using knock out cell lines deficient for key RNAi factors including proteins of the argonaute (AGO) and trinucleotide repeat containing protein 6 (TNRC6) families. We will combine mass spectrometry, RNAseq, and cross linking immunoprecipitation (CLIP) to define where interactions occur how the number of protein or miRNAs affect the impact on gene regulation. We will first examine detailed mechanisms for regulation in cell cytoplasm. We will then apply these lessons to investigate the consequences of miRNA recognition in both cell cytoplasm and nuclei. Novel mechanisms and disease targets. We will gain a better understanding of two diseases caused by mutant RNAs, Fuch's Corneal Endothelial Dystrophy (FECD) and Friedreich's Ataxia (FA). One goal is to develop more potent compounds to facilitate drug development. A second goal is to better understand mechanisms of action. Both diseases involve nuclear noncoding RNAs, and we will use synthetic nucleic acids as tools to probe disease mechanism and better appreciate how a small number of mutant RNA molecules can cause late-onset degenerative diseases.