OptoRibo-seq for spatiotemporally resolved mapping of local protein translatome

Localized protein translation occurs in numerous subcellular compartments and regulates diverse biological processes by rapidly changing protein compositions in response to subcellular needs. Existing assays for subcellular local translation either require physical isolation, which is prone to contamination and loss of material, or imaging-based readout, which is often hampered with low throughput. In this study, we report the development of optoRibo-seq method that features photoactivatable enzyme-mediated proximity labeling of ribosomes in genetically specified subcellular locations. We demonstrate the spatial specificity of optoRibo-seq at the endoplasmic reticulum (ER) membrane and mitochondrial outer membrane. OptoRibo-seq is further applied to map the dynamic changes in the ER-proximal translatome in cells undergoing chemically induced ER stress, identifying transcripts involved in protein folding and amino acid transport. Our strategy provides a general platform for spatiotemporally resolved profiling of subcellular protein translation in various organelles.