School of Biological Sciences, University of Southampton, Southampton, Hampshire SO17 1BJ, UK.
Centre for Human Development, Stem Cells and Regeneration, University of Southampton, Southampton, Hampshire SO17 1BJ, UK.
Nucleic Acids Res. 2020 Sep 25;48(17):9822-9839. doi: 10.1093/nar/gkaa699.
RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5' UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.
RNA 四链体（G4s）是一种被提出的二级结构，作为调节转录后 mRNA 定位和翻译的功能体。已知一些神经元 mRNA 中的 G4s 控制远端定位和局部翻译，有助于形成独特的局部蛋白质组，从而促进正常细胞功能所必需的突触重塑。在这项研究中，我们对钾离子 2 孔域渗漏通道 Task3 mRNA 5'UTR 内发现的（GGN）13 重复序列的 G4 形成进行了表征。生物物理分析表明，该（GGN）13 重复序列在体外形成平行 G4，表现出 G4 的典型钾离子特异性，在生理离子条件下保持热稳定性。通过小鼠脑组织 G4-RNA 免疫沉淀，我们进一步证实了 Task3 mRNA 在体内形成 G4 结构。该 G4 抑制了体外的 Task3 翻译，但通过 G4 特异性解旋酶 DHX36 的活性得到克服，增加了 HEK293 细胞中的 K+渗漏电流和膜超极化。此外，我们观察到这种 G4 对于确保 Task3 mRNA 递送到远端初级皮质神经元突起是至关重要的。已经表明，异常的 Task3 表达与神经元功能障碍相关，因此我们假设这种 G4 对于调节 Task3 渗漏通道的局部表达至关重要，这些通道维持神经元内的 K+渗漏。
