ORFRATER analysis of ribosome profiling data from primary mouse cortical neurons

Learning and memory require activity-induced changes in mRNA translation within dendrites, but which mRNAs are involved and how they are regulated remain unclear. We combined proximity labeling with ribosome profiling and CLIP to monitor how depolarization impacts dendritic translation. For a functionally coherent set of transcripts highly enriched in mitochondrial genes, depolarization leads to enhanced uORF translation, eIF4G2 binding, and increased translation. Engineered reporters demonstrate that activity-dependent translational control is conferred by the 5'UTRs and that dendritic localization, eIF4G2 binding, and uORF translation are necessary and sufficient to mediate this regulation. Downstream, this drives activity-dependent changes in dendritic mitochondrial function. Our studies uncover an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 enables the coupling of synaptic activity to local remodeling of dendrites. Overall design: Three 150 mm cell culture dishes were combined per sample. Neurons were either treated with nothing (WT), cycloheximide for 2 minutes (Chx), or harringtonine for 2 minutes followed by a Chx pulse. Cells were quickly rinsed in ice-cold polysome gradient buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 µg/ml CHX), then scraped and lysed on plates on ice in 1 mL of ice-cold polysome lysis buffer (20 mM Tris pH7.5, 150 mM NaCl, 5 mM MgCl2 supplemented with 20 U/mL SUPERase-In RNase inhibitor, 24 U/mL Turbo DNase, 1 mM DTT, and 100 µg/ml CHX, 1x EDTA-free protease inhibitor. Lysates were clarified by centrifugation at 20,000 g for 2 minutes at 4°C and subsequently flash-frozen or used for monosome fractionation. For monosome fractionation, lysates were added 5 mM CaCl2, incubated with micrococcal nuclease for 45 minutes at room temperature, quenched with 6.25 mM EGTA, and loaded on sucrose gradients. Gradients were then centrifuged for 2 hours at 41K rpm in an SW-41 rotor, and monosomes were collected using the BioComp Gilson fraction collection.