Department of Chemistry, Marquette University, Milwaukee 53233, WI, United States.
Department of Biochemistry, Vanderbilt University Medical Center, Nashville 37205-0146, TN, United States.
J Struct Biol. 2021 Jun;213(2):107728. doi: 10.1016/j.jsb.2021.107728. Epub 2021 Mar 20.
HOTAIR is a large, multi-exon spliced non-coding RNA proposed to function as a molecular scaffold and competes with chromatin to bind to histone modification enzymes. Previous sequence analysis and biochemical experiments identified potential conserved regions and characterized the full length HOTAIR secondary structure. Here, we examine the thermodynamic folding properties and structural propensity of the individual exonic regions of HOTAIR using an array of biophysical methods and NMR spectroscopy. We demonstrate that different exons of HOTAIR contain variable degrees of heterogeneity, and identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing unambiguously confirm conserved base pair interactions within the structural element, termed helix 10 of exon 4, located within domain I of human HOTAIR. This combined exon-biased and integrated biophysical approach introduces a new strategy to examine conformational heterogeneity in lncRNAs and emphasizes NMR as a key method to validate base pair interactions and corroborate large RNA secondary structures.
HOTAIR 是一个大型的、多外显子拼接的非编码 RNA，被认为具有分子支架的功能，并与染色质竞争，以结合组蛋白修饰酶。先前的序列分析和生化实验鉴定了潜在的保守区域，并对全长 HOTAIR 的二级结构进行了特征描述。在这里，我们使用一系列生物物理方法和 NMR 光谱法研究了 HOTAIR 各个外显子区域的热力学折叠特性和结构倾向。我们证明 HOTAIR 的不同外显子具有不同程度的异质性，并鉴定出一个外显子区域，即外显子 4，在低镁浓度下会形成稳定紧凑的折叠。NMR 光谱和化学探测的紧密一致性，明确地证实了位于人 HOTAIR 结构域 I 内的结构元件（称为外显子 4 的第 10 螺旋）内保守的碱基对相互作用。这种组合的exon 偏向和综合生物物理方法为研究 lncRNA 中的构象异质性引入了一种新策略，并强调 NMR 是验证碱基对相互作用和证实大 RNA 二级结构的关键方法。
