Biophysical investigations of RNA complexes essential for gene expression

基因表达必需的 RNA 复合物的生物物理学研究

• 批准号: 9071523
• 负责人: Samuel E Butcher
• 金额: $ 8.86万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071523
• 关键词: Acute; Affinity; Antiviral Therapy; Binding; Biological Models; Biological Process; Cell Nucleolus; Cells; Code; Complex; Development; Essential Genes; Event; Gene Expression; Genetic Transcription; Genomics; HIV; HIV-1; Health; Human; Internal Ribosome Entry Site; Investigation; Lead; Link; Measures; Mediating; Messenger RNA; Molecular; Paralysed; Play; Process; Proteins; RNA; RNA Splicing; Reading Frames; Research; Resolution; Retroviridae; Ribonucleases; Ribonucleoproteins; Ribosomal Frameshifting; Ribosomal RNA; Ribosomes; Role; Site; Spliceosome Assembly Pathway; Spliceosomes; Structure; Testing; Thermodynamics; Time; Untranslated RNA; Viral; Virus; Virus Replication; angiogenin; base; biophysical techniques; blood vessel development; in vivo; mRNA Precursor; novel; particle; programs; public health relevance; small molecule; tool

 DESCRIPTION (provided by applicant): RNA molecules play myriad essential roles in gene expression, yet for many cellular and viral processes, little is known about how RNA complexes coordinate and regulate these dynamic events. This project will apply biophysical approaches aimed at revealing how RNA interactions drive important biological processes such as pre-mRNA splicing and translational frameshifting. Pre-mRNA splicing is catalyzed by the spliceosome, a large and highly dynamic assembly of 5 RNAs and over 100 proteins. We will investigate how spliceosomal RNAs and their associated complexes interact during spliceosome assembly, a process involving large-scale RNA structural rearrangements that ultimately lead to the formation of the spliceosome, a massive ribonucleoprotein particle twice the size of a ribosome. There are no high-resolution structures of the spliceosome, and very little is known at the molecular level about the steps leading to its assembly and activation. Translational frameshifting is another RNA-mediated process that we aim to study and for which there is no high-resolution structural information available. Frameshifting is the process by which ribosomes are directed into an alternate reading frame to synthesize a different protein. Most retroviruses utilize translational frameshifting in the form of an RNA programmed -1 frameshift, which increases the viral genomic coding capacity and serves to regulate the expression of essential genes. This proposal will examine HIV-1 frameshifting and will measure for the first time the relationship between HIV-1 mRNA thermodynamic stability and frameshift efficiency in vivo. We will also explore how frameshifting is linked to RNA packaging in HIV and will test novel, high affinity compounds that bind to the HIV-1 frameshift site, stimulate frameshifting and inhibit HIV replication. Using the tools we have developed for HIV, we will expand these investigations to study the structural basis for +1 reading frame selection, using the well-studied Israeli Acute Paralysis Virus internal ribosome entry site as a model system. These studies will significantly advance our understanding of how mRNAs program translational recoding in human cells, and may eventually lead to the development of novel antiviral therapies. Finally, we will capitalize on a recent breakthrough to explore an exciting new direction aimed at understanding how angiogenin stimulates formation of blood vessels. Angiogenin is a ribonuclease that has been recently found to specifically bind to a non-coding RNA in the nucleolus in order to activate transcription of rRNA, the first step in inducing cellula proliferation.

 描述（由申请人提供）：RNA 分子在基因表达中发挥着无数重要作用，但对于许多细胞和病毒过程，人们对 RNA 复合物如何协调和调节这些动态事件知之甚少。该项目将应用生物物理学方法，旨在揭示 RNA 相互作用如何驱动重要的生物过程，例如前 mRNA 剪接和翻译移码。 mRNA 前体剪接由剪接体催化，剪接体是由 5 个 RNA 和 100 多种蛋白质组成的大型且高度动态的组装体。我们将研究剪接体 RNA 及其相关复合物在剪接体组装过程中如何相互作用，剪接体组装是一个涉及大规模 RNA 结构重排的过程，最终导致剪接体的形成，剪接体是一种两倍于核糖体大小的巨大核糖核蛋白颗粒。剪接体没有高分辨率的结构，并且在分子水平上对导致其组装和激活的步骤知之甚少。翻译移码是我们旨在研究的另一种 RNA 介导的过程，但目前还没有可用的高分辨率结构信息。移码是将核糖体引导至替代阅读框以合成不同蛋白质的过程。大多数逆转录病毒利用RNA编程-1移码形式的翻译移码，这增加了病毒基因组编码能力并用于调节必需基因的表达。该提案将研究 HIV-1 移码，并首次测量 HIV-1 mRNA 热力学稳定性和体内移码效率之间的关系。我们还将探索移码如何与 HIV 中的 RNA 包装联系起来，并将测试与 HIV-1 移码位点结合、刺激移码并抑制 HIV 复制的新型高亲和力化合物。利用我们为 HIV 开发的工具，我们将使用经过充分研究的以色列急性麻痹病毒内部核糖体进入位点作为模型系统，扩大这些研究范围，以研究 +1 阅读框选择的结构基础。这些研究将显着增进我们对 mRNA 如何在人类细胞中编程翻译重新编码的理解，并可能最终导致新型抗病毒疗法的开发。最后，我们将利用最近的突破来探索一个令人兴奋的新方向，旨在了解血管生成素如何刺激血管形成。血管生成素是一种核糖核酸酶，最近发现它可以特异性结合核仁中的非编码RNA，以激活rRNA的转录，这是诱导细胞增殖的第一步。