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

Abstract

Cells regulate protein synthesis in response to fluctuating nutrient availability through coordinated mechanisms that affect both translation initiation and elongation. Branched-chain amino acids (BCAAs)--leucine, isoleucine, and valine--are essential nutrients with a significant impact on cellular physiology. However, how their simultaneous depletion affects translation remains largely unclear. Here, we examined the immediate effects of short-term BCAA limitation on translational dynamics in mammalian cells. We performed RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) on NIH3T3 cells subjected to single, double, or triple BCAA deprivation. Our analyses revealed increased ribosome dwell times in all starvation conditions, with pronounced stalling at all valine codons during valine and triple starvation, while leucine and isoleucine starvation produced milder, codon-specific effects. Notably, we could show that isoleucine starvation-specific stalling largely diminished under triple starvation, likely due to early elongation bottlenecks at valine codons. Correlating these stalling events with tRNA charging levels revealed distinct tRNA isoacceptor regulation in each starvation condition. In addition, integrating quantitative proteomics showed that many proteins downregulated under BCAA deprivation harbor stalling sites, suggesting that compromised elongation contributes to decreased protein output. Together, these findings suggest that differential ribosome stalling under BCAA starvation reflects a balance between amino acid supply, tRNA charging dynamics, and stress-response signaling, illustrating that codon choice shapes the translational landscape under nutrient limitation.