A subcellular map of translational machinery composition and regulation at the single-molecule level [ALIBi_Lsg1_KD]

Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. Here, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing unexpected subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Finally, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution. Rpl31-FLAG-TEV-AviTag-containing ribosomes were proximity biotinylated using ALIBi targeted to either the cytosol or endoplasmic reticulum. Biotinylated ribosomes were affinity purified and subjected to poly-A-enriched RNA-seq. Whole-cell RNA from the same cell lysates (input for affinity purification) was sequenced in parallel. In some cases, cells were treated with either a targeting or scrambled (control) siRNA prior to performing ALIBi-mediated enrichment of subcellular ribosome populations.