Abstract
Translation is a central control point of gene expression, linking nucleotide sequences to functional proteins. Dysregulated translation contributes to diverse diseases, underscoring the need for methods that can directly reveal which transcripts are actively translated. Ribosome profiling, the current gold standard, provides nucleotide-resolution maps of ribosome occupancy but requires laborious purification and sacrifices information on mRNA isoforms and mRNA modifications by restricting analysis to short ribosome-protected fragments. Here, we introduce Protein Synthesis Profiling (PSP), a proximity-labeling strategy for transcriptome-wide identification of actively translated mRNAs without ribosome isolation. PSP exploits a fusion of the enzyme APEX2 with the elongation factor eEF2, which transiently associates with ribosomes during elongation, to catalyze selective tagging of mRNAs engaged in translation. Applied in Saccharomyces cerevisiae, PSP captures condition-specific translational programs, recapitulates known stress responses, and expands the detectable repertoire of regulated genes beyond existing methods. By preserving full-length transcript features, PSP is scalable, isoform-aware, and broadly adaptable, providing a versatile platform to dissect translational regulation in health and disease.