Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons

•Here we describe neurite/soma separation scheme applied to mESC-derived neurons.•Neurite/soma separation is combined with mass spectrometry, RNAseq and bioinformatics analyses.•The method enables identification of localized and locally translated proteins and RNAs. The subcellular localization and...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2019-06, Vol.162-163, p.31-41
Main Authors: Ludwik, Katarzyna A., von Kuegelgen, Nicolai, Chekulaeva, Marina
Format: Article
Language:English
Subjects:
Local translation
MRNA localization
Neuron
Non-coding RNAs
RNA-binding proteins (RBPs)
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Summary:•Here we describe neurite/soma separation scheme applied to mESC-derived neurons.•Neurite/soma separation is combined with mass spectrometry, RNAseq and bioinformatics analyses.•The method enables identification of localized and locally translated proteins and RNAs. The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments – cell bodies (soma) and cell extensions (axons and dendrites) – for further biochemical analyses. Here we describe neurite/soma separation approach in combination with RNA sequencing and proteomic analyses to identify localized and locally translated RNAs and proteins. This approach allows quantification of around 7000 of local proteins and the entire local transcriptome. It provides a powerful tool for investigation of the mechanisms underlying RNA localization and local translation in neurons.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2019.02.002