eIF4G2 cross-linking immunoprecipitation (CLIP) in primary 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. Neurons were washed twice with 1x PBS with 100 μg/ml cycloheximide (CHX) and UV-crosslinked on ice in the same wash buffer with one pulse of 400 mJ/cm2 and one pulse of 200 mJ/cm2. Cells were then immediately scraped in fresh 1x ice-cold PBS with CHX and centrifuged at 7500 rpm for 5 minutes at 4°C. The pellets were flash frozen or processed as described previously for established CLIP protocols with modifications. Three biological replicates were used, each replicate prepared from three 15-cm cell culture dishes. Pellets were resuspended in 0.5 m lysis buffer, and immunoprecipitations (IP) were performed overnight at 4°C using the rabbit monoclonal antibody against eIF4G2.