Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA; National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, USA.
J Mol Biol. 2023 Dec 15;435(24):168340. doi: 10.1016/j.jmb.2023.168340. Epub 2023 Nov 2.
Poly(UG) or "pUG" RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3' ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU), as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or "pUG fold." Overall, the structure agrees with the crystal structure of (GU) bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.
聚（UG）或“pUG”RNAs 是富含真核生物的 UG 或 GU 二核苷酸重复序列。pUG 在 RNA 3' 末端的转录后添加将 mRNAs 标记为线虫中基因沉默的载体。我们之前确定了与配体 N-甲基 mesoporphyrin IX（NMM）结合的 pUG RNA 的晶体结构，但游离 pUG RNA 的结构尚不清楚。在这里，我们通过核磁共振波谱和小角和广角 X 射线散射（NMR-SAXS-WAXS）报告了游离 pUG RNA（GU）的溶液结构。该结构的低复杂度序列和 4 倍对称性导致重叠的 NMR 信号，这使得化学位移分配复杂化。因此，我们利用了单个位点特异性脱氧核糖修饰，这些修饰不会破坏结构，并引入了在 NMR 光谱中易于识别的分辨率良好的亚甲基信号。溶液结构组合的均方根偏差（RMSD）为 0.62 Å，是一种紧凑的左手四重构象，具有 Z 型骨架，或“pUG 折叠”。总体而言，该结构与与 NMM 结合的（GU）的晶体结构一致，表明 pUG 折叠在与 NMM 配体对接时不会改变。溶液结构揭示了无法通过 X 射线晶体学解决的构象细节，这些细节解释了 pUG 折叠如何在更长的 RNA 内形成。
