Division of Endocrinology and Metabolism, National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, Sichuan, China.
Division of Respiratory and Critical Care Medicine, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, Sichuan, China.
J Hepatol. 2020 Aug;73(2):371-382. doi: 10.1016/j.jhep.2020.02.032. Epub 2020 Mar 10.
BACKGROUND & AIMS: RNA G-quadruplexes (RG4s) appear to be important in post-transcriptional gene regulation, but their pathophysiological functions remain unknown. MicroRNA-26a (miR-26a) is emerging as a therapeutic target for various human diseases, however the mechanisms underlying endogenous miR-26a regulation are poorly understood. Herein, we study the role of RG4 in miR-26a expression and function in vitro and in vivo.
Putative RG4s within liver-enriched miRNAs were predicted by bioinformatic analysis, and the presence of an RG4 structure in the miR-26a-1 precursor (pre-miR-26a-1) was further analyzed by biophysical and biochemical methods. RG4 stabilizers, pre-miR-26a-1 overexpression plasmids, and luciferase reporter assays were used to assess the effect of RG4 on pre-miR-26a-1 maturation. Both miR-26a knock-in and knockout mouse models were employed to investigate the influence of this RG4 on miR-26a expression and function. Moreover, the interaction between RG4 in pre-miR-26a-1 and DEAH-box helicase 36 (DHX36) was determined by biophysical and molecular methods. Finally, miR-26a processing and DHX36 expression were quantified in the livers of obese mice.
We identify a guanine-rich sequence in pre-miR-26a-1 that can fold into an RG4 structure. This RG4 impairs pre-miR-26a-1 maturation, resulting in a decrease in miR-26a expression and subsequently an increase in miR-26a cognate targets. In line with known miR-26a functions, this RG4 can regulate hepatic insulin sensitivity and lipid metabolism in vitro and in vivo. Furthermore, we reveal that DHX36 can bind and unwind this RG4 structure, thereby enhancing miR-26a maturation. Intriguingly, there is a concordant decrease of miR-26a maturation and DHX36 expression in obese mouse livers.
Our findings define a dynamic DHX36/RG4/miR-26a regulatory axis during obesity, highlighting an important role of RG4 in physiology and pathology.
Specific RNA sequences called G-quadruplexes (or RG4) appear to be important in post-transcriptional gene regulation. Obesity leads to the formation of these RG4 structures in pre-miR-26a-1 molecules, impairing the maturation and function of miR-26a, which has emerged as a therapeutic target in several diseases. This contributes to hepatic insulin resistance and the dysregulation of liver metabolism.
RNA 四链体（RG4）似乎在转录后基因调控中起着重要作用，但它们的病理生理功能尚不清楚。MicroRNA-26a（miR-26a）作为各种人类疾病的治疗靶点正在出现，然而，内源性 miR-26a 调节的机制尚不清楚。在此，我们研究了 RG4 在 miR-26a 在体外和体内表达和功能中的作用。
通过生物信息学分析预测富含肝脏的 miRNA 中的假定 RG4，并通过生物物理和生化方法进一步分析 miR-26a-1 前体（pre-miR-26a-1）中 RG4 结构的存在。使用 RG4 稳定剂、pre-miR-26a-1 过表达质粒和荧光素酶报告基因检测来评估 RG4 对 pre-miR-26a-1 成熟的影响。使用 miR-26a 敲入和敲除小鼠模型来研究该 RG4 对 miR-26a 表达和功能的影响。此外，通过生物物理和分子方法确定 pre-miR-26a-1 中的 RG4 与 DEAH-box 解旋酶 36（DHX36）之间的相互作用。最后，在肥胖小鼠的肝脏中定量检测 miR-26a 加工和 DHX36 表达。
我们在 pre-miR-26a-1 中鉴定出一个可以折叠成 RG4 结构的富含鸟嘌呤的序列。该 RG4 会损害 pre-miR-26a-1 的成熟，导致 miR-26a 表达减少，随后 miR-26a 同源靶标增加。与已知的 miR-26a 功能一致，该 RG4 可在体外和体内调节肝脏胰岛素敏感性和脂质代谢。此外，我们揭示了 DHX36 可以结合并解开这种 RG4 结构，从而增强 miR-26a 的成熟。有趣的是，肥胖小鼠肝脏中 miR-26a 成熟和 DHX36 表达都有相应下降。
我们的研究结果定义了肥胖过程中动态的 DHX36/RG4/miR-26a 调控轴，突出了 RG4 在生理和病理中的重要作用。
称为 G-四链体（或 RG4）的特定 RNA 序列似乎在转录后基因调控中起着重要作用。肥胖导致 pre-miR-26a-1 分子中形成这些 RG4 结构，损害 miR-26a 的成熟和功能，miR-26a 作为几种疾病的治疗靶点正在出现。这导致肝脏胰岛素抵抗和肝脏代谢失调。
