Transfer RNA acetylation regulates in vivo mammalian stress signaling [tissues]

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here, we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification catalyzed by the essential acetyltransferase Nat10. By targeting Thumpd1, a nonessential adapter protein required for Nat10-catalyzed tRNA acetylation, we determine that loss of tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2a phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality in mice, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translational control and therapeutic interventions. Overall design: RNA-Seq samples from mouse tissues in which Thumpd1 gene has been knocked out (Thumpd1 KO) vs control (WT).

转运核糖核酸（Transfer RNA, tRNA）修饰在蛋白质合成过程中发挥关键作用，但其位点特异性的生理功能仍未得到充分解析。本研究针对由必需乙酰转移酶Nat10催化的高度保守tRNA修饰——N4-乙酰胞苷（N4-acetylcytidine, ac4C）的影响展开探究。我们通过靶向Thumpd1——一种介导Nat10催化tRNA乙酰化过程的非必需衔接蛋白——发现，tRNA乙酰化缺失会导致亮氨酸转运RNA（tRNALeu）水平降低、核糖体停滞现象增多，并激活eIF2α磷酸化通路。Thumpd1基因敲除小鼠会表现出生长缺陷与不育表型。同时敲除Thumpd1与应激感应激酶Gcn2会导致小鼠出现完全外显的产后致死表型，这表明二者之间存在关键的遗传互作关系。本研究结果表明，仅局限于II型胞质转运RNA单个位点的修饰，即可调控哺乳动物体内核糖体介导的应激信号通路，这对于我们理解翻译调控机制以及开发治疗干预手段具有重要参考价值。实验整体设计：以Thumpd1基因敲除（Thumpd1 KO）小鼠的组织RNA测序样本与野生型（WT）对照样本为比较对象。