Activity-dependent ribosome profiling reveals the landscape of canonical and non-canonical translation in brain tissue [RIP-Seq]

Neural activity-dependent translation is essential for synaptic plasticity and diverse brain functions. Translation involves not only canonical main open reading frames (mORFs) but also upstream ORFs (uORFs), which may regulate mORF expression. However, due to technical limitations, systematic in-vestigation of activity-dependent uORFs and mORFs in brain tissues remains challenging. Here, we developed a ribosome tagging and purification strategy that bypasses the prolonged turnover of ribo-somal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation. Applying this strategy to mouse hippocampal slices undergoing long-term potentiation, we identified hundreds of activity-induced mORFs and uORFs, a subset of which served as robust markers for ac-tivity status. Notably, ~22% of the upregulated uORFs overlapped with those induced by the integrated stress response, suggesting potential crosstalk between these signaling pathways. This study provides a useful technique and resources for deciphering molecular mechanisms underlying activity- and trans-lation-dependent brain functions in health and disease. Overall design: RNA-seq, P5 mice Organotypic hippocampus slice culture, EGFP-L10a, Ha-GFPnanobody-HALO-PEST