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.
Highlights
The emergence of the system approach to the study of neuron function came from the realization that no protein or process can function in isolation but is often embedded in a network of regulating interactions
Intracerebellar injection of AAV2/2-8 at P4 resulted in intense expression of the EYFP-RPL10A construct in up to eight lobules of the vermis (Fig. 1B), variable spread to lateral hemisphere (Supplemental Fig. S1), and expression restricted to Purkinje cells (PC), both in mice and rat (Fig. 1B–E)
Since RPL10A itself was found to be present in Purkinje dendrites, we used it as the ribosome-targeting component of the probe
Summary
The emergence of the system approach to the study of neuron function came from the realization that no protein or process can function in isolation but is often embedded in a network of regulating interactions. The cataloging of building parts is further complicated by its dynamic nature, with protein concentration being modified through transcriptional and post-transcriptional regulation, as well as local destruction or synthesis of components These modifications are functionally important because protein synthesis in general and especially local synthesis in dendrites are required for synapse maturation and plasticity (Martin and Ephrussi 2009; Liu-Yesucevitz et al 2011). Quantification of translating mRNA is expected to be a better proxy measurement of protein synthesis (Schwanh€ausser et al 2011) than the total mRNA level, which has long been recognized as a poor predictor of protein abundance (Gygi et al 1999) The use of both TRAP and RiboTag is practically limited to mice since these strategies require transgenic animals engineered to express a modified ribosomal protein (RPL10 for TRAP and RPL22 for RiboTag). Since the available RNA was limited in quantity and the genome of the chosen model animal, Rattus norvegicus is not annotated as extensively as for mouse or human, we chose the high-sensitivity paired-end nanoCAGE/CAGEscan implementation of CAGE (Plessy et al 2010) to identify TSS independently of existing annotation
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