Schlundt Andreas, Tants Jan-Niklas, Sattler Michael
Center for Integrated Protein Science Munich (CiPSM) at the Department of Chemistry, Technische Universität München, 85748 Garching, Germany; Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
Center for Integrated Protein Science Munich (CiPSM) at the Department of Chemistry, Technische Universität München, 85748 Garching, Germany; Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
Methods. 2017 Apr 15;118-119:119-136. doi: 10.1016/j.ymeth.2017.03.015. Epub 2017 Mar 16.
Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain arrangements and dynamics. Principles of protein-RNA recognition and current approaches are reviewed and illustrated with recent studies.
RNA测序技术的最新进展极大地扩展了我们对细胞中RNA景观的认识，而且常常具有时空分辨率。这些技术鉴定出了许多新的（通常是非编码的）RNA分子。大规模研究还发现了新型RNA结合蛋白（RBP），它们具有单个或多个RNA结合结构域（RBD），用于识别RNA中的特定序列或结构化基序。从这些大规模方法出发，揭示核糖核蛋白复合物（RNP）中蛋白质-RNA识别的分子原理对于理解基因调控的潜在机制至关重要。尽可能高分辨率的结构生物学和生物物理研究是阐明RBP识别RNA的分子机制以及构象动力学、弱相互作用和协同结合如何促成特定的、依赖于上下文的RNP形成的关键。虽然大型紧密RNP可以通过X射线晶体学和冷冻电镜进行很好的研究，但对动力学和弱相互作用的分析需要使用溶液方法来捕捉这些特性。在这里，我们阐述了从鉴定给定RNP中的相互作用伙伴开始，研究溶液中蛋白质-RNA复合物的结构和构象动力学的方法。生物物理和生化技术支持对蛋白质-RNA复合物的表征，并识别与结构分析相关的区域。核磁共振（NMR）是获取有关RNA折叠、稳定性和动力学信息以及表征溶液中RNP的强大工具。它提供了与其他技术得出的静态图像互补的关键信息。NMR可以很容易地与其他溶液技术相结合，如小角X射线和/或中子散射（SAXS/SANS）、电子顺磁共振（EPR）和荧光共振能量转移（FRET），这些技术提供有关整体形状、内部结构域排列和动力学的信息。本文回顾了蛋白质-RNA识别的原理和当前方法，并通过近期研究进行了说明。
