Cornilescu Gabriel, Didychuk Allison L, Rodgers Margaret L, Michael Lauren A, Burke Jordan E, Montemayor Eric J, Hoskins Aaron A, Butcher Samuel E
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
J Mol Biol. 2016 Feb 27;428(5 Pt A):777-789. doi: 10.1016/j.jmb.2015.11.026. Epub 2015 Dec 2.
NMR and SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) are highly complementary approaches for the analysis of RNA structure in solution. Here we describe an efficient NMR-SAXS/WAXS approach for structural investigation of multi-helical RNAs. We illustrate this approach by determining the overall fold of a 92-nt 3-helix junction from the U4/U6 di-snRNA. The U4/U6 di-snRNA is conserved in eukaryotes and is part of the U4/U6.U5 tri-snRNP, a large ribonucleoprotein complex that comprises a major subunit of the assembled spliceosome. Helical orientations can be determined by X-ray scattering data alone, but the addition of NMR RDC (residual dipolar coupling) restraints improves the structure models. RDCs were measured in two different external alignment media and also by magnetic susceptibility anisotropy. The resulting alignment tensors are collinear, which is a previously noted problem for nucleic acids. Including WAXS data in the calculations produces models with significantly better fits to the scattering data. In solution, the U4/U6 di-snRNA forms a 3-helix junction with a planar Y-shaped structure and has no detectable tertiary interactions. Single-molecule Förster resonance energy transfer data support the observed topology. A comparison with the recently determined cryo-electron microscopy structure of the U4/U6.U5 tri-snRNP illustrates how proteins scaffold the RNA and dramatically alter the geometry of the U4/U6 3-helix junction.
核磁共振（NMR）和小角X射线散射（SAXS）/广角X射线散射（WAXS）是用于分析溶液中RNA结构的高度互补的方法。在此，我们描述了一种用于多螺旋RNA结构研究的高效NMR-SAXS/WAXS方法。我们通过确定来自U4/U6双小核RNA的92个核苷酸的三螺旋连接体的整体折叠来阐明这种方法。U4/U6双小核RNA在真核生物中保守，并且是U4/U6.U5三小核核糖核蛋白复合体的一部分，该复合体是一种大型核糖核蛋白复合体，包含组装的剪接体的一个主要亚基。螺旋取向可以仅通过X射线散射数据确定，但是添加NMR剩余偶极耦合（RDC）约束可改善结构模型。在两种不同的外部取向介质中以及通过磁化率各向异性测量了RDC。所得的取向张量是共线的，这是核酸之前已注意到的问题。在计算中包括WAXS数据会产生与散射数据拟合度明显更好的模型。在溶液中，U4/U6双小核RNA形成具有平面Y形结构的三螺旋连接体，并且没有可检测到的三级相互作用。单分子荧光共振能量转移数据支持所观察到的拓扑结构。与最近确定的U4/U6.U5三小核核糖核蛋白复合体的冷冻电子显微镜结构进行比较，说明了蛋白质如何构建RNA支架并显著改变U4/U6三螺旋连接体的几何形状。
