Li Fangyuan, Guo Dan, Xie Ting, Zhang Sumei, Wang Anqi, Li YingXing, Zhou Jiang
Medical Research Central, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, China; Clinical Biobank, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, China.
Medical Research Central, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, China; Clinical Biobank, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, China.
Int J Biol Macromol. 2023 Mar 15;231:123279. doi: 10.1016/j.ijbiomac.2023.123279. Epub 2023 Jan 16.
A certain proportion of pre-miRNAs, which contained potential G-quadruplex forming sequences, was found to act as a mediator to Dicer-mediated cleavage, and that the regulation of miRNA production and function may be achieved through the G-quadruplex structure. In this study, human precursor miR-1587 sequence was transfected after the incubation with different solution conditions (K, TMPyP4, etc.). Firstly, the formation of G-quadruplex from precursor miR-1587 sequences was confirmed by CD and UV melting. The expression of miR-1587 level was then evaluated by Q-RT-PCR, and the results showed that the formation of G-quadruplex inhibited the miR-1587 maturation process, resulting in a reduced miR-1587 expression. Meanwhile the destabilization of G-quadruplex led to an increased miR-1587 expression by contrast. Then, the weakened inhibition of miR-1587 towards its target genes, such as TAGLN or NCOR1, was presented confirming by Q-RT-PCR and western blot. Molecular mechanism by dual-luciferase assays showed that the modulations of miR-1587 expression and function were due to the G-quadruplex structure transformation, but not the simple change of solution conditions. This study highlighted the importance of maintaining specific structures during miRNA biosynthesis and provided a way to alter the function of G-rich precursor miRNAs by modulating molecular conformation using ionic solutions or ligands.
研究发现，一定比例的含有潜在G-四链体形成序列的前体微小RNA（pre-miRNA）可作为Dicer介导的切割的介质，并且微小RNA的产生和功能的调节可能通过G-四链体结构来实现。在本研究中，人源前体miR-1587序列在与不同溶液条件（钾离子、TMPyP4等）孵育后进行转染。首先，通过圆二色光谱（CD）和紫外熔解实验证实了前体miR-1587序列形成了G-四链体。然后通过定量逆转录聚合酶链反应（Q-RT-PCR）评估miR-1587的表达水平，结果表明G-四链体的形成抑制了miR-1587的成熟过程，导致miR-1587表达降低。同时，相比之下，G-四链体的去稳定化导致miR-1587表达增加。随后，通过Q-RT-PCR和蛋白质免疫印迹法证实了miR-1587对其靶基因（如TAGLN或NCOR1）的抑制作用减弱。双荧光素酶报告基因实验的分子机制表明，miR-1587表达和功能的调节是由于G-四链体结构的转变，而不是溶液条件的简单变化。本研究强调了在微小RNA生物合成过程中维持特定结构的重要性，并提供了一种通过使用离子溶液或配体调节分子构象来改变富含鸟嘌呤的前体微小RNA功能的方法。
