ABSTRACT

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m¹G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

摘要

RNA 含有多种与其碱基或糖基共价连接的转录后修饰。这些修饰的核苷由于 RNA 分解代谢而从 RNA 分子中释放出来，并释放到细胞外空间，但细胞外运输的分子机制及其病理生理学意义尚不清楚。在本研究中，我们发现RNA衍生的修饰核苷通过平衡核苷转运蛋白1和2（ENT1和ENT2）输出到细胞外空间，ENT1比ENT2对修饰核苷表现出更高的偏好。ENT1和ENT2的药理抑制或基因缺失显着减弱了修饰核苷的输出，从而导致它们在细胞质中积累。使用诱变策略，我们鉴定了 ENT1 中参与区分未修饰和修饰核苷的氨基酸残基。在 ENTs 缺陷细胞中，细胞内修饰核苷水平升高与自噬反应的诱导密切相关，LC3-II 水平升高证明了这一点。重要的是，我们对能够诱导自噬的修饰核苷进行了筛选，发现 1-甲基鸟苷 (m ¹ G) 足以诱导 LC3-II 水平。从病理生理学角度来看，修饰核苷的缺陷输出会以自噬依赖性方式显着诱导寨卡病毒复制。此外，我们还发现地拉西普对ENTs的药理学抑制显着诱导寨卡病毒复制。总的来说，我们的研究结果强调了 RNA 衍生的修饰核苷作为激活自噬反应的重要信号调节剂，并表明这些修饰核苷的输出缺陷可能对病理生理学产生深远的影响。