Conjunction of G-quadruplex and stem-loop in the 5' untranslated region of mouse hepatocyte nuclear factor 4-alpha1 mediates strong inhibition of protein expression.
Conjunction of G-quadruplex and stem-loop in the 5' untranslated region of mouse hepatocyte nuclear factor 4-alpha1 mediates strong inhibition of protein expression.
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DOI：
10.1007/s11010-018-3274-3
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发表时间：
2018-09
影响因子：
4.3
通讯作者：
Lu H
中科院分区：
文献类型：
--
作者：
Guo S;Lu H
肝细胞核因子 4-α (HNF4α) 是肝脏发育和功能的公认主要调节因子。恢复 HNF4α 可以治疗多种肝脏疾病和肝癌。迄今为止，由于缺乏已知的激活配体，HNF4α 仍然是“不可成药的”。因此，了解 HNF4α 表达的调节机制可能有助于开发一种调节 HNF4α 蛋白水平的替代方法。 G-四链体 (G4) 是非典型的稳定二级结构，主要发现于癌基因的启动子中。最近的全基因组研究表明 G4 在 5` 非翻译区 (UTR) 中富集。通过原配蛋白-IX 结合测定和圆二色光谱，我们验证了小鼠 Hnf4a1 5'UTR 内化学高度稳定的 4 环 G4 的存在。我们的实时 PCR 和蛋白质印迹数据表明，尽管对 Hnf4a1 mRNA 水平有中等影响，但 Hnf4a1 5`UTR 引起了显着的翻译抑制。随后使用双荧光素酶报告基因测定对 Hnf4a1 5' UTR 进行的缺失/突变分析进一步证明，尽管化学上高度稳定的 4 环 G4 的破坏导致抑制作用显着减弱，但单独的 G4 仅微弱地抑制翻译。同样，破坏与 4 环 G4 相邻的长茎环会显着减弱翻译抑制，尽管单独的茎环仅发挥微弱的抑制作用。因此，G4 和 Hnf4a1 5`UTR 内相邻茎环的紧密结合对于介导非常强的翻译抑制是必要且充分的。我们的结果建立了一个新的工作模型，化学稳定的 G4 可能需要辅因子具有生物稳定性才能发挥生物功能。
Hepatocyte nuclear factor 4-alpha (HNF4α) is a well-established master regulator of liver development and function. Restoration of HNF4α can treat multiple liver disorders and liver cancers. To date, HNF4α is still “undruggable” due to lack of known activating ligands. Thus, understanding the regulatory mechanism of HNF4α expression may help develop an alternative approach to modulate HNF4α protein levels. G-quadruplexes (G4) are non-canonical stable secondary structures discovered mostly in the promoters of oncogenes. Recent genome-wide studies demonstrate the enrichment of G4s in the 5` untranslated region (UTR). By protophrin-IX binding assay and circular dichroism spectrum, we validated the presence of a chemically highly stable 4-ring G4 within the 5`UTR of mouse Hnf4a1. Our real-time PCR and Western blot data showed that the Hnf4a1 5`UTR caused a remarkable translational suppression regardless of a moderate effect on Hnf4a1 mRNA levels. The subsequent deletion/mutation analysis of Hnf4a1 5` UTR using dual-luciferase reporter assays further demonstrated that although the disruption of the chemically highly stable 4-ring G4 resulted in a marked attenuation of inhibition, the G4 alone only weakly inhibited translation. Likewise, disruption of a long stem-loop adjacent to the 4-ring G4 markedly attenuated translational inhibition, although the stem-loop alone only exerted a weak inhibitory effect. Thus, the tight conjunction of G4s and an adjacent stem-loop within the Hnf4a1 5` UTR was both necessary and sufficient to mediate the very strong translational repression. Our results establish a novel working model that a chemically stable G4 may require co-factors to be bio-stable for exerting biological functions.
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