Distinct translational profiles reveal elongation bottlenecks and codon-specific stalling during branched-chain amino acid deprivation [Ribo-Seq]

ABSTRACT: Cells regulate protein synthesis in response to fluctuating nutrient availability through highly 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 the translational machinery and dynamics remains largely unclear. Here, we examine 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 the deprived conditions, with especially strong stalling at all valine codons during valine and triple starvation, while leucine and isoleucine depletion triggered more moderate, codon-specific effects. Notably, isoleucine-specific stalling largely diminished under triple deprivation, 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 proteome data 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 limitation reflects a balance between amino acid supply, tRNA charging dynamics, and stress pathway modulation, illustrating that codon usage biases can profoundly influence the global landscape of translation under nutrient stress.

ORGANISM(S): Mus musculus

PROVIDER: GSE291653 | GEO | 2025/09/02

REPOSITORIES: GEO