Dysregulation of amino acid metabolism upon rapid depletion of cap-binding protein eIF4E [pcl5_screen]

Protein synthesis is metabolically costly, and the level of translation must match nutrient availability and cellular needs. Overall protein synthesis levels are modulated by regulating translation initiation. The cap-binding protein eIF4E—the earliest contact between mRNAs and the translation machinery—serves as one point of control, but its contributions to mRNA-specific translation regulation remain poorly understood. We acutely depleted eIF4E, which is essential in budding yeast, and observed surprisingly modest effects on cell growth and protein synthesis. Long-lived transcripts were downregulated, likely reflecting accelerated turnover, and the strongest gene-specific effects arose as secondary effects of reduced protein biosynthesis on amino acid pools. Futile cycles of amino acid synthesis and degradation were accompanied by translational activation of GCN4, which is typically induced by amino acid starvation. We further identified translational tuning of PCL5, a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varying translation environments. This translational control depended in part on a uniquely long poly-(A) tract in the PCL5 5’ UTR and on poly-(A) binding protein. These results highlight the intricate interplay between translation, amino acid homeostasis, and gene regulation and uncover new layers of feedback control in cellular response to stress and nutrient availability.蛋白质合成在代谢层面成本高昂，翻译水平必须与养分可用性及细胞需求相匹配。整体蛋白质合成水平通过调控翻译起始过程实现调节。帽结合蛋白eIF4E（cap-binding protein eIF4E）——mRNA与翻译机器间的首个接触位点——是诸多调控节点之一，但其在mRNA特异性翻译调控中的具体贡献仍未得到充分阐明。我们在出芽酵母中急性耗竭了必需蛋白eIF4E，意外观察到其对细胞生长与蛋白质合成的影响极为有限。长寿命转录本的表达出现下调，这大概率反映了转录本降解的加速；而最显著的基因特异性效应，源于蛋白质生物合成减弱对氨基酸库产生的次级影响。氨基酸合成与降解的无效循环，伴随通常在氨基酸饥饿时被诱导的GCN4的翻译激活。我们进一步发现了PCL5的翻译调控机制：PCL5作为Gcn4的负调控因子，可在不同翻译环境下维持稳定的蛋白质与mRNA比例。该翻译调控一定程度上依赖于PCL5 5'非翻译区（5' UTR）中一段异常冗长的聚腺苷酸（poly-(A)）序列，以及聚腺苷酸结合蛋白。本研究结果揭示了翻译、氨基酸稳态与基因调控间的复杂互作，并阐明了细胞应对胁迫与养分可用性时反馈调控的全新层级。