Digital expression profiling of the compartmentalized translatome of Purkinje neurons
- Anton Kratz1,5,6,
- Pascal Beguin2,6,
- Megumi Kaneko2,
- Takahiko Chimura2,3,
- Ana Maria Suzuki1,5,
- Atsuko Matsunaga2,
- Sachi Kato1,5,
- Nicolas Bertin1,4,5,
- Timo Lassmann1,5,
- Réjan Vigot2,
- Piero Carninci1,5,
- Charles Plessy1,5 and
- Thomas Launey2
- 1RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, 230-0045 Japan;
- 2RIKEN Brain Science Institute, Launey Research Unit, Wako, Saitama, 351-0198 Japan
- Corresponding authors: plessy{at}riken.jp, thomas.launey{at}riken.jp
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↵6 These authors contributed equally to this work.
Abstract
Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions, and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons pose a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome—the translatome—from selected subcellular domains of specific neurons, and apply it to the Purkinje cells (PCs) in the rat cerebellum. We combined microdissection, translating ribosome affinity purification (TRAP) in nontransgenic animals, and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)–associated ribosomes in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities.
Footnotes
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↵5 These members of CLST belonged to RIKEN OSC before the RIKEN reorganization of April 1, 2013.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.164095.113.
Freely available online through the Genome Research Open Access option.
- Received August 26, 2013.
- Accepted April 29, 2014.
This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0.