In-Cell Discovery of Functional RNA Tertiary Structures

RNA分子在细胞中扮演着特殊的角色，因为它们既编码组成RNA链的线性碱基序列(缩写为A、C、G和U)中的信息，也通过形成链内相互作用来编码信息。这些链内相互作用导致主要由Watson-Crick碱基配对稳定的“二级”结构，现在许多情况下都可以很容易地测量到，以及产生复杂而紧凑的三维形状的“三级”结构，这一直很难测量。然而，测量三级结构很重要，因为它们影响细胞中RNA分子的功能，包括蛋白质的合成，调节特定基因合成多少蛋白质，以及其他重要任务。直到最近，几乎不可能检测到大多数RNA的三级结构，特别是在活细胞中。通过这个项目，将创造一种新的技术来识别RNA三级结构的位置。在这个项目的背景下，这项技术将被用来理解RNA分子的三级结构如何影响它们在人类细胞中的功能。这个项目还包括本科生研究人员，目的是激励这些学生了解研究如何创造知识，并丰富我们对细胞过程的理解，这两者对科学进步、经济发展、技术创新和生活质量都很重要。新的RNA口袋将通过一个完全由本科生驱动的研究努力来探索，本科生转录组项目。参与这项研究的本科生和研究生都有望在STEM领域取得行业和学术界的领导地位。这项研究项目的智力价值和总体愿景是创建一种严格的、易于实施的技术，专门检测活细胞中大RNA中真正的高阶三级结构。尽管很明显，许多RNA形成复杂的结构，并且这些结构对细胞功能有重要的影响，但只有少数已知的不同类别的基序形成真正的三级结构。通过在活细胞中发现RNA三级结构，该项目将定义RNA分子的许多新功能。这项研究项目将使用最近开发的一种化学试剂，该试剂专门检测RNA分子中的高阶三级相互作用。这些构象只出现在多个RNA螺旋聚集在一起创建电负性RNA口袋的位置。这些罕见但具有高度诊断性的位点可以通过带正电荷的小试剂有效地检测出来，揭示出第三级相互作用的位点(称为T-位点)。这项技术将被用来检查活细胞中人类转录组中的T位点，并了解T位点与翻译调控之间存在关系的初步发现。这项技术将先进到可以被非专家广泛使用的程度。预计这项工作的结果将是多方面的。首先，预计T位点与基因调控的连接点有很强的相关性。其次，将发现许多新的RNA基序类别，大大扩展了目前已知的重要但相对较少的RNA三级结构类别。第三，后续的生物物理和功能研究将支持关于RNA结构和折叠的基本原理及其对调节基因表达的过程的影响的许多新发现。该项目由生物科学局分子和细胞生物科学部的分子生物物理学和遗传机制群支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

RNA molecules play special roles in cells because they encode information both in the linear sequence of nucleobases (abbreviated A, C, G, and U) that make up an RNA strand and by forming intra-strand interactions. These intra-strand interactions result in "secondary" structures stabilized primarily by Watson-Crick base pairing, which can now be readily measured in many cases, and "tertiary" structures that result in complex and compact three-dimensional shapes, which have been very difficult to measure. Measuring tertiary structures is important, however, as they affect how RNA molecules function in cells, including in synthesis of proteins, in regulation of how much protein is made from a particular gene, and in other important tasks. Until recently, it was nearly impossible to detect most RNA tertiary structures, especially in living cells. Through this project, a new technology will be created to identify sites of RNA tertiary structures. Within the context of this project, this technology will be used to understand how tertiary structures in RNA molecules influence their functions in human cells. This project also incorporates undergraduate researchers with the goal of inspiring these students to become informed about how research creates knowledge and enriches our understanding of cellular processes, both of which are important for scientific advances, economic development, and innovations in technology and quality of life. Novel RNA pockets will be explored through a completely undergraduate-driven research endeavor, the Undergraduate Transcriptome Project. Both undergraduate and graduate students who participate in this research are expected to achieve leadership roles in STEM fields in industry and academics. The Intellectual Merit and overarching vision of this research project is to create a rigorous, easily implemented technology that specifically detects true higher order tertiary structures in large RNAs in living cells. Although it is clear that many RNAs form complex structures and that these structures have important consequences for cellular function, there are only a handful of known distinct classes of motifs that form true tertiary structures. By discovering RNA tertiary structures in living cells, this project will define numerous new functions of RNA molecules. This research project will use a recently developed chemical reagent that specifically detects higher order tertiary interactions in RNA molecules. These conformations only occur at sites where multiple RNA helices pack together to create an electronegative RNA pocket. These rare but highly diagnostic sites are detected efficiently by the small, positively charged reagent, revealing sites of tertiary interactions (termed T-sites). This technology will be used to examine T-sites across the human transcriptome in living cells and to understand a preliminary finding that relationships exist between T-sites and translational regulation. The technology will be advanced to the point where it can be widely used by non-experts. Results of this work are expected to be multi-fold. First, T-sites are expected to be strongly correlated with nexuses of gene regulation. Second, many new classes of RNA motifs will be discovered, substantially extending the important, but relatively few, classes of RNA tertiary structures currently known. Third, subsequent biophysical and functional studies will support numerous new discoveries regarding the fundamental principles of RNA structure and folding and their impacts on processes that regulate gene expression. This project is supported by the Molecular Biophysics and Genetic Mechanism Clusters of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.