show Abstracthide AbstractActivity-dependent protein synthesis is critical for determining changes in dendritic proteomes underlying brain function, yet the mechanisms governing these changes are lacking. Here, we combined proximity-based labeling of dendritic transcriptome, translatome, and proteome to study the dynamics of RNA regulation in activated synapses. We discovered that depolarization leads to a switch from RNAs translated under basal conditions to new translation of previously unrecognized subsets of depolarization-dependent transcripts. Dynamically regulated RNAs bound by specific regulatory proteins, including NOVA1, FMRP, and ELAVLs, rapidly generate proteins with roles in mitochondrial regulation, RNA metabolism, translational control, and synaptic signaling in dendrites. Knockdowns of activity-induced dendritic RNAs altered neuronal physiology, underscoring how dynamic switches in the regulation of RNAs encoding coordinated sets of proteins underlie synaptic plasticity. Overall design: Four 150 mm cell culture dishes for resting and depolarized primary cortical neurons were combined per sample per replicate, 1/4th of which was spared for proteomics and rest for the ribosome profiling experiments. After a two-minute cycloheximide (Chx) treatment followed by a 30-minute biotin 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, after which the supernatant was immediately loaded on to the cold 5 mL 7 kDa MWCO Zeba desalting column that was previously equilibrated with the ice-cold polysome gradient buffer. Lysates were 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 spun for 2 hours at 41K rpm in an SW-41 rotor and fractionated using BioComp Gilson fraction collection. 10% of the monosome fraction was kept for input. Triton X-100 was added to the remaining monosomes to a final concentration of 0.05%. The biotinylated monosomes were isolated using 50 µl of MyOne streptavidin C1 magnetic Dynabeads that were prepared according to the manufacturer's guidelines and washed twice with Buffer A, twice with Buffer B, and once with polysome gradient buffer (with DTT and CHX). The monosome and bead mix was nutated at 4°C overnight. The supernatant was removed the next day, and the beads were moved into a new tube with low salt wash buffer (20 mM Tris pH8, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 µg/ml CHX, and 0.1% Triton X-100), and consequently washed three times for 10 minutes at 4°C with high salt wash buffer (20 mM Tris pH8, 500 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml CHX, and 0.1% Triton X-100).