Activity-dependent ribosome profiling reveals the landscape of canonical and non-canonical translation in brain tissue
TL;DR
A ribosome tagging and purification strategy is developed that bypasses the prolonged turnover of ribosomal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation, and uncovered previously unknown activity-dependent alternative translation with functional implication, such as the uORF of Egr1.
Abstract
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 investigation 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 ribosomal proteins, enabling ribosome profiling with one-hour temporal resolution after neural stimulation. Applying this strategy to mouse hippocampal slices undergoing long-term potentiation, we identify hundreds of activity-induced mORFs and uORFs, including a previously unknown uORF from Egr1. We demonstrate that this Egr1-uORF translation is tightly regulated by neuronal activity, and its encoded peptide interacts with peroxisomal machinery, suggesting a potential link between synaptic stimulus and peroxisome biology. This study provides a useful technique and resources for deciphering molecular mechanisms underlying activity- and translation-dependent brain functions in health and disease. Investigation of activity-dependent protein synthesis selectively in activated neurons has long been a challenge. Here, the authors developed a technique to avoid the translational background from inactive neurons and uncovered previously unknown activity-dependent alternative translation with functional implication, such as the uORF of Egr1.