Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity.

Quantifying gene expression in space, for example by spatial transcriptomics, is essential for describing the biology of cells and their interactions in complex tissues. Perturbation experiments, at single-cell resolution and conditional on both space and time, are necessary for dissecting the molecular mechanisms of these interactions. To this aim, we combined optogenetics and CRISPR technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatial patterns. As a proof of principle, we optogenetically induced Sonic Hedgehog (SHH) signaling at a distinct spatial location within human neural organoids. This robustly induced known SHH spatial domains of gene expression, cell-autonomously and across the entire organoid. In principle, our approach can be used to induce or knock down RNAs from any gene of interest in specific spatial locations or patterns of complex biological systems.