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（由适用提供）：RNA分子在基因表达中起着无数的基本作用，但是对于许多细胞和病毒过程，关于RNA复合物如何坐标和调节这些动态事件的情况知之甚少。该项目将采用旨在揭示RNA相互作用如何驱动重要生物学过程（例如MRNA剪接和翻译Fameshifting）的生物物理方法。前MRNA剪接是由剪接体催化的，这是5个RNA和超过100种蛋白质的大型且高度动态的组装。我们将研究剪接体RNA及其相关复合物如何在剪接体组装过程中相互作用，该过程涉及大规模RNA结构重排的过程，最终导致剪接体形成，这是核糖体大小的两倍。没有剪接体的高分辨率结构，在分子水平上几乎不知道导致其组装和激活的步骤。转化帧汇总是我们旨在研究的另一个RNA介导的过程，并且没有可用的高分辨率结构信息。框架是将核糖体引导到替代阅读框中以合成不同蛋白质的过程。大多数逆转录病毒以RNA编程的-1帧速率的形式利用翻译帧，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。该提案将检查HIV-1帧速率，并将首次测量HIV-1 mRNA热力学稳定性与体内移码效率之间的关系。我们还将探讨如何与HIV中的RNA包装相关联，并将测试与HIV-1 Frameshift位点结合的新颖的高亲和力化合物，刺激Frameshifting并抑制HIV复制。使用我们为艾滋病毒开发的工具，我们将使用精心研究的以色列急性瘫痪病毒内部核糖体进入站点作为模型系统来扩展这些投资，以研究+1阅读框架选择的结构基础。这些研究将大大提高我们对人类细胞中mRNA程序如何翻译的理解，并最终可能导致新型抗病毒疗法的发展。最后，我们将利用最近的突破，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的形成。血管生成蛋白是一种核糖核酸酶，最近发现它特异性结合了核仁中的非编码RNA，以激活rRNA的转录，这是诱导的细胞增殖的第一步。