mRNA stability and m6A are major determinants of subcellular mRNA localization in neurons. Loedige et al.
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Original Images to Publication: “mRNA stability and m6A are major determinants of subcellular mRNA localization". For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called “zipcodes” that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here we assess the role of mRNA stability in localization by combining isolation of the soma and neurites of primary cortical and mESC-derived neurons, SLAM-seq, m6A-RIP-seq, perturbation of mRNA destabilization mechanisms, and analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m6A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons.
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2E) Detection of newly synthesized proteins by Puro-PLA was performed on mouse PCNs cultured on 15-mm glass coverslips. Briefly, PCNs were infected with 8 µl concentrated lentiviral particles (shRNA against Larp1 or non-targeting control shRNA) on DIV3. On DIV10, neurons were incubated with 1 mg/ml puromycin for 15 min, washed quickly in PBS and fixed with 4% PFA in PBS for 10 min at RT. Cells were washed twice in PBS and permeabilized with 0.2 % Triton X-100 in PBS for 10 min at RT. Puro-PLA was performed using Duolink reagent, antibodies α-actin-ß (1/50) and α-puromycin (1/200) and rabbit PLAplus and mouse PLAminus probes according to manufacturer’s recommendations except that antibody dilution solution was replaced by 5 % BSA in PBS. Neurons were immunostained with α-MAP2 (1/500) and α-chicken-alexa 488 (1/1000) and mounted in Duolink in situ mounting medium. Images were acquired using a 40x oil objective on a Leica SP8 FALCON confocal microscope. Z-stacks (0.3 µm) were chosen to span the entire volume of the neurite but not the entire soma. 4C) For m6A dot blot, indicated amount of RNA was pipetted on a nitrocellulose membrane fixed between the plates of a dot blot apparatus and UV-crosslinked twice with 1200 uJ (auto crosslink mode). The membrane was washed in PBST (PBS, 0.1% tween-20), blocked in PBST with 5% non-fat milk for 30 min and probed with anti-m6A antibody 1:1000 (Synaptic Systems 202111) in PBST with 5% non-fat milk.