Although recent advances in sequencing technology have enabled unprecedented study of gene expression, systematic investigation of its sub-cellular and tissue-level heterogeneity remains a major challenge. With regard to protein expression, translational activity is known to vary in space at different subcellular sites, with ribosome composition, and over a translational cycle through interactions with mRNA or ribosome-associated factors. Here, I describe the development, validation, and application of a genetically controllable proximity-specific ribosome profiling approach to monitor the translational activity of sub-pools of ribosomes in vivo. Importantly, it is both comprehensive and highly precise, enabling mechanistic study of the spatiotemporal dynamics of translation at a genome scale.

We apply the this approach to study local translation at two broadly conserved subcellular sites, the endoplasmic reticulum (ER) and mitochondria, and also demonstrate its feasibility for specifically isolating ribosomes which interact with soluble factors. Study of ER translation revealed that co-translational translocation is far more pervasive than was previously thought, and that the location of a signal sequence within a protein is the major determinant for undergoing this form of import. Additionally, dissecting the functional roles of distinct translocon complexes enabled a more sophisticated understanding of how the cell supports efficient co-translational import of a diverse set of substrates. Interrogation of local mRNA translation at the mitochondrial outer membrane highlights additional questions whose study is enabled by proximity-specific ribosome profiling. We obtained direct evidence for co-translational import of mitochondrial inner membrane proteins in vivo, resolving a decades-long debate and in the field regarding the mechanism of protein insertion. By exploiting the sensitivity of the approach, we confidently identified dozens of unannotated candidate mitochondrial proteins. Furthermore, we demonstrate the utility of our method for systematic study of protein dual localization through synthesis of our ER and mitochondrial datasets. The specificity of these spatially resolved proteomic maps revealed a novel ER-form of fumarate reductase, suggesting a mechanism for oxidative folding in the ER under anaerobic conditions. At a unique interface of gene expression and cell biology, future applications of proximity-specific ribosome profiling will inform our understanding of translational heterogeneity within cells and tissues.