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

 描述（由申请人提供）：我的实验室研究天然和化学修饰的核酸的性质。在细胞生物学和核酸化学的界面上工作，我们已经改变了细胞活动，包括转录、翻译、蛋白质表达的等位基因选择性抑制、剪接和端粒聚合酶介导的端粒延长。2016-2020年，我们的中心目标是研究细胞RNA的识别，主要关注核RNA。我们将把这种理解应用于控制基因表达的方法和对基因调控的自然途径的新见解。基因组的大部分被转录成不编码蛋白质的RNA。许多非编码RNA的功能尚不清楚，它们如何影响基因表达的机制也不清楚。已发表的报告对特定非编码RNA如何影响给定基因表达的分子细节几乎没有提供任何见解。在缺乏这些信息的情况下，验证所提出的效应变得有问题，并且研究新基因的预测能力有限。缺乏指导性的机械原则是取得进展的主要障碍。 RNAi提供了识别特定核RNA种类的潜在机制。虽然RNAi作为识别哺乳动物细胞质中mRNA的驱动力是众所周知的，但其驱动体细胞核中识别的潜力已经被证实。 不清楚。我们建议定义哺乳动物核RNAi的范围和机制，并将其应用于新的疾病靶点。 目的1.了解核RNAi。我们将描述核RNAi中涉及的蛋白质和RNA相互作用。RNAseq将用于鉴定RNA靶标以进行功能验证。质谱法将用于确定蛋白质伴侣。我们将探索核RNAi的机制，并深入了解核RNAi如何调节哺乳动物细胞中的基因表达。这些数据将揭示argonaute蛋白和其他RNAi因子如何在细胞核中发挥作用以控制基因表达。 目标2.细胞内核酸的新靶点。我们还将扩大对新的核酸靶点的识别，包括突变体frataxin和C9 orf 72基因内的扩展内含子重复序列。这些数据将进一步发展我们对核RNAi机制的理解，并显示核RNAi如何应用于疾病基因表达的控制。控制共济失调蛋白表达或破坏内含子C9 orf 72内扩展重复序列形成的结构的化合物将是药物开发的先导化合物。

 DESCRIPTION (provided by applicant): My laboratory investigates the properties of natural and chemically modified nucleic acids. Working at the interface of cell biology and nucleic acid chemistry we have modified cellular activities including transcription, translation, allele-selectie inhibition of protein expression, splicing, and telomerase-mediated elongation of telomeres. For 2016-2020 our central goal is to examine the recognition of cellular RNA with a primary focus on nuclear RNA. We will apply that understanding to methods for controlling gene expression and new insights into natural pathways of gene regulation. Much of the genome is transcribed into RNAs that do not encode proteins. The function of many noncoding RNAs is unclear, as is the mechanism for how they might affect gene expression. Published reports offer little insight into the molecular details for how a specific noncoding RNA affects expression of a given gene. In the absence of this information, validation of proposed effects becomes problematic and the predictive power for studying novel genes is limited. This lack of guiding mechanistic principles is a major obstacle to progress. RNAi provides a potential mechanism for recognizing specific nuclear RNA species. While RNAi is well-known as a driving force for recognition of mRNA in the cytoplasm of mammalian cells, its potential to drive recognition in the somatic cell nuclei has been unclear. We propose to define the scope and mechanism of mammalian nuclear RNAi and to apply it to novel disease targets. Objective 1. Understand nuclear RNAi. We will characterize the protein and RNA interactions involved in nuclear RNAi. RNAseq will be used to identify RNA targets for functional validation. Mass spectrometry will be used to determine protein partners. We will explore the mechanism of nuclear RNAi and obtain insights into the how nuclear RNAi regulates gene expression in mammalian cells. These data will reveal how argonaute proteins and other RNAi factors function in the nucleus to control gene expression. Objective 2. Novel targets for nucleic acids inside cells. We will also expand recognition to novel nucleic acids targets including the expanded intronic repeats within the mutant frataxin and C9orf72 genes. These data will further develop our understanding of the mechanism of nuclear RNAi and show how nuclear RNAi can be applied to the control of disease gene expression. Compounds that control frataxin protein expression or disrupt structures formed by the expanded repeat within intronic C9orf72 would be lead compounds for drug development.