Codon-specific ribosome stalling reshapes translational dynamics during branched-chain amino acid starvation

Background: Cells regulate protein synthesis in response to fluctuating nutrient availability through mechanisms that affect both translation initiation and elongation. Branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients. However, how their depletion affects translation remains largely unclear. Here, we investigate the immediate effects of single, double, and triple branched-chain amino acid deprivation on translational dynamics in NIH3T3 cells using RNA-seq and ribosome profiling. Results: All starvation conditions increased ribosome dwell times, with pronounced stalling at all valine codons during valine and triple starvation, whereas leucine and isoleucine starvation produced milder, codon-specific effects. Notably, stalling under isoleucine deprivation largely decreased under triple starvation. Positional enrichment of valine codons near the 5' end and downstream isoleucine codons potentially contributes to these patterns, suggesting a possible elongation bottleneck that influences translational responses under branched-chain amino acid starvation. The presence of multiple valine stalling sites was associated with decreased protein levels. Finally, codon-specific dwell time changes correlated strongly with patterns of tRNA isoacceptor charging. Conclusions: Together, these findings suggest that differential ribosome stalling under branched-chain amino acid starvation reflects a balance between amino acid supply, tRNA charging dynamics, codon position, and stress-response signaling. Overall design: NIH3T3 mouse fibroblast cells were grown either in complete medium (control) or in medium lacking specific branched-chain amino acids for 6 hours.

背景：细胞可通过影响翻译起始与延伸的多种机制，响应营养供给波动以调控蛋白质合成。支链氨基酸（branched-chain amino acids），即亮氨酸、异亮氨酸与缬氨酸，均为必需营养素。然而，这类氨基酸的匮乏如何影响蛋白质翻译，目前仍未得到充分阐明。本研究采用RNA测序（RNA-seq）与核糖体谱（ribosome profiling）技术，探究单种、两种、三种支链氨基酸剥夺对NIH3T3细胞翻译动态的即时影响。
结果：所有饥饿处理组均延长了核糖体驻留时间；在缬氨酸饥饿与三种支链氨基酸饥饿条件下，所有缬氨酸密码子位点均出现显著的核糖体停滞，而亮氨酸饥饿与异亮氨酸饥饿仅引发较弱的密码子特异性效应。值得注意的是，异亮氨酸匮乏条件下的核糖体停滞现象，在三种支链氨基酸饥饿时大幅减弱。5'端附近的缬氨酸密码子与下游异亮氨酸密码子的位置富集特征，或可解释上述观测模式，提示存在一类翻译延伸瓶颈，可影响支链氨基酸饥饿状态下的细胞翻译应答。多个缬氨酸停滞位点的存在与蛋白质表达水平下调显著相关。最后，密码子特异性的核糖体驻留时间变化，与tRNA同工受体（tRNA isoacceptor）的氨酰化负载模式显著相关。
结论：综上，本研究结果表明，支链氨基酸饥饿诱导的差异性核糖体停滞，反映了氨基酸供给水平、tRNA氨酰化动态、密码子位置与应激应答信号通路之间的动态平衡。
实验整体设计：将NIH3T3小鼠成纤维细胞培养于完全培养基（对照组）或缺失特定支链氨基酸的培养基中，处理时长为6小时。