In-Cell Discovery of Functional RNA Tertiary Structures

RNA分子在细胞中扮演着特殊的角色，因为它们在构成RNA链的核碱基线性序列（缩写为A、C、G和U）和形成链内相互作用中编码信息。这些链内相互作用导致主要由沃森-克里克碱基配对稳定的“二级”结构，现在在许多情况下可以很容易地测量，而“三级”结构导致复杂而紧凑的三维形状，这是非常难以测量的。然而，测量三级结构是很重要的，因为它们影响RNA分子在细胞中的功能，包括蛋白质的合成，调节由特定基因产生的蛋白质数量，以及其他重要任务。直到最近，几乎不可能检测到大多数RNA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，这项技术将被用来了解RNA分子的三级结构如何影响它们在人类细胞中的功能。该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，这两者对于科学进步，经济发展以及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工业和学术界的STEM领域发挥领导作用。这个研究项目的智力价值和总体愿景是创造一种严格的、易于实施的技术，专门检测活细胞中大型rna中真正的高阶三级结构。虽然很明显，许多rna形成复杂的结构，这些结构对细胞功能有重要影响，但只有少数已知的不同类型的基序形成真正的三级结构。通过发现活细胞中的RNA三级结构，该项目将定义RNA分子的许多新功能。该研究项目将使用一种最近开发的化学试剂，专门检测RNA分子中的高阶三级相互作用。这些构象只发生在多个RNA螺旋聚集在一起形成电负性RNA口袋的地方。这些罕见但具有高度诊断性的位点被小的带正电的试剂有效地检测到，揭示了三级相互作用的位点（称为t位点）。这项技术将用于检查活细胞中人类转录组中的t位点，并了解t位点与翻译调节之间存在关系的初步发现。这项技术将发展到可以被非专业人士广泛使用的程度。这项工作的结果预计将是多方面的。首先，t位点被认为与基因调控的联系密切相关。其次，许多新的RNA基序将被发现，大大扩展了目前已知的重要但相对较少的RNA三级结构。第三，后续的生物物理和功能研究将支持许多关于RNA结构和折叠的基本原理及其对调节基因表达过程的影响的新发现。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学和遗传机制集群支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.