Department of Chemistry & Biochemistry, University of the Sciences, Philadelphia, PA 19143, USA.
Nucleic Acids Res. 2019 Feb 20;47(3):1468-1481. doi: 10.1093/nar/gky1171.
Nucleic acid triplexes may regulate many important biological processes. Persistent accumulation of the oncogenic 7-kb long noncoding RNA MALAT1 is dependent on an unusually long intramolecular triple helix. This triplex structure is positioned within a conserved ENE (element for nuclear expression) motif at the lncRNA 3' terminus and protects the entire transcript from degradation in a polyA-independent manner. A requisite 3' maturation step leads to triplex formation though the precise mechanism of triplex folding remains unclear. Furthermore, the contributions of several peripheral structural elements to triplex formation and protective function have not been determined. We evaluated the stability, conformational fluctuations, and function of this MALAT1 ENE triple helix (M1TH) protective element using in vitro mutational analyses coupled with biochemical and biophysical characterizations. Using fluorescence and UV melts, FRET, and an exonucleolytic decay assay we define a concerted mechanism for triplex formation and uncover a metastable, dynamic triplex population under near-physiological conditions. Structural elements surrounding the triplex regulate the dynamic M1TH conformational variability, but increased triplex dynamics lead to M1TH degradation. Taken together, we suggest that finely tuned dynamics may be a general mechanism regulating triplex-mediated functions.
核酸三聚体可能调节许多重要的生物过程。致癌的 7kb 长非编码 RNA MALAT1 的持续积累依赖于异常长的分子内三聚体。这种三聚体结构位于 lncRNA 3'末端的保守 ENE（核表达元件）基序内，并以多聚 A 非依赖性的方式保护整个转录本免受降解。一个必需的 3'成熟步骤导致三聚体形成，尽管三聚体折叠的确切机制仍不清楚。此外，几个外围结构元件对三聚体形成和保护功能的贡献尚未确定。我们使用体外突变分析结合生化和生物物理特性评估了 MALAT1 ENE 三聚体（M1TH）保护元件的稳定性、构象波动和功能。通过荧光和 UV 熔融、FRET 和外切核酸酶降解分析，我们定义了三聚体形成的协同机制，并在接近生理条件下揭示了一种亚稳定、动态的三聚体群体。三聚体周围的结构元件调节动态 M1TH 构象变异性，但增加的三聚体动力学导致 M1TH 降解。总之，我们认为精细调节的动力学可能是调节三聚体介导功能的一般机制。
