Biophysical investigations of RNA complexes essential for gene expression

 描述（由申请人提供）：RNA分子在基因表达中起着无数重要作用，但对于许多细胞和病毒过程，关于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方法，旨在揭示RNA相互作用如何驱动重要的生物过程，如前mRNA剪接和翻译移码。前体mRNA剪接由剪接体催化，剪接体是5种RNA和100多种蛋白质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，剪接体是一种巨大的核糖核蛋白颗粒，大小是核糖体的两倍。剪接体没有高分辨率的结构，在分子水平上对导致其组装和激活的步骤知之甚少。翻译移码是另一个RNA介导的过程，我们的目标是研究和没有高分辨率的结构信息。移码是指核糖体被引导到另一个阅读框架中以合成不同蛋白质的过程。大多数逆转录病毒以RNA程序化-1移码的形式利用翻译移码，其增加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，并将首次测量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移码位点结合的新型高亲和力化合物，刺激移码并抑制HIV复制。使用我们为HIV开发的工具，我们将扩展这些研究，以研究+1阅读框选择的结构基础，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。这些研究将大大推进我们对mRNA如何在人类细胞中编程翻译重编码的理解，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，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。血管生成素是一种核糖核酸酶，最近发现其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。

 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.