Spatially Resolved Single-cell Translatomics at Molecular Resolution

The precise control of mRNA translation is a crucial step in post-transcriptional gene regulation of cellular physiology. However, it remains a major challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of RIBOmap, a three-dimensional (3D) in situ profiling method to detect mRNA translation of thousands of genes simultaneously in intact cells and tissues. By applying RIBOmap to 981 genes in HeLa cells, we revealed a remarkable dependency of translation on cell-cycle stages and subcellular localization. Furthermore, we profiled single-cell translatomes of 5,413 genes in adult mouse brain tissues yielding a spatial cell atlas of 119,173 cells. The pairwise spatial mapping of single-cell translatome and transcriptome in two adjacent mouse brain slices revealed cell-type and brain-region-dependent translational regulation and suggested a translation remodeling during oligodendrocyte lineage maturation. The spatial translatome profiling detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks. Together, RIBOmap presents the first spatially resolved single-cell translatomics technology, accelerating our understanding of protein synthesis in the context of subcellular architecture, cell types, and tissue anatomy.