Summary
The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation. Among these, alternative splicing has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell (sc) resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with long-read scRNA-sequencing (scRNA-seq) in the brain. We generated the first isoform-sensitive sc translatomes of the mouse hippocampus at postnatal day 25, discovering cell type-specific translation of 3,857 alternative transcripts across 1,641 genes, and identifying isoforms of the same genes undergoing differential translation within and across eight different cell types. We defined high and low translational states in CA1 and CA3 neurons, with synaptic and metabolic genes enriched in high states. Surprisingly, we found that CA3 exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of translational machinery components. This accessible platform will expand our understanding of how cell type- and isoform-specific translation drives brain physiology and disease.