Abstract
Neurons rely on translation of synaptic mRNAs in order to generate activity-dependent changes in plasticity. Here, we develop a strategy combining compartment-specific crosslinking immunoprecipitation (CLIP) and translating ribosome affinity purification (TRAP) in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. We identify CA1 dendritic transcripts with differentially localized mRNA isoforms generated by alternative polyadenylation and alternative splicing, including many that have altered protein-coding capacity. Among dendritic mRNAs, FMRP targets were found to be overrepresented. Cell-type-specific FMRP-CLIP and TRAP in microdissected CA1 neuropil revealed 383 dendritic FMRP targets and suggests that FMRP differentially regulates functionally distinct modules in CA1 dendrites and cell bodies. FMRP regulates ~15-20% of mRNAs encoding synaptic functions and 10% of chromatin modulators, in the dendrite and cell body, respectively. In the absence of FMRP, dendritic FMRP targets had increased ribosome association, consistent with a function for FMRP in synaptic translational repression. Conversely, downregulation of FMRP targets involved in chromatin regulation in cell bodies suggests a role for FMRP in stabilizing mRNAs containing stalled ribosomes in this compartment. Together, the data support a model in which FMRP regulates the translation and expression of synaptic and nuclear proteins within different compartments of a single neuronal cell type.
Highlights
A key feature in the molecular biology of learning and memory is protein-synthesis dependent synaptic plasticity, which involves translation of localized mRNAs in response to synaptic activity
Crossing these animals with Camk2a-Cre mice results in lines expressing tagged ribosomes, or in the case of conditionally tagged 102 (cTag) polyA-binding protein c1 (PABPC1) or FMRP, “knock-in” tagged proteins expressed from native genes. This expression is specific in the CA1 pyramidal neurons (Figure 1 – supplement 1A; Hwang et al, 2017). 134 Microdissection of the CA1 neuropil compartments and immunoprecipitation (IP) allowed us to enrich 135 dendritic tagged proteins that originated from the cell bodies (CB) of the CA1 neurons
We identified two groups of dendritic, ribosome-bound mRNAs: dendrite-present and dendrite-enriched. 689 (34%) of the dendrite-present mRNAs were previously identified in bulk RNA-seq of the microdissected rat CA1 neuropil (Cajigas et al., 2012), and these RNAs were found to be significantly enriched in dendrites (CA1 neuropil TRAP vs bulk RNA-seq; Figure 1 – supplement 2A-B)
Summary
A key feature in the molecular biology of learning and memory is protein-synthesis dependent synaptic plasticity, which involves translation of localized mRNAs in response to synaptic activity. FMRP target genes overlap significantly with autism susceptibility genes and include genes involved in both synaptic function and transcriptional control in the nucleus (Darnell, 2020; Darnell et al, 2011; Iossifov et al, 2012; Sawicka et al, 2019), and loss of FMRP increases translation of chromatin modifiers such as BRD4 (Korb et al, 2017) and SETD2 (Shah et al, 2020) These and other observations have suggested a model in which FMRP regulates the stoichiometry of its targets in two ways: globally, by translational control of transcription regulators in the cell body, and locally, by enabling activity-dependent local translation of synaptic proteins in dendrites (Darnell, 2020), but it is still unclear the extent to which such regulation occurs simultaneously in a single neuron. Together these findings support a model in which distinct patterns of both mRNA and FMRP subcellular localization enable FMRP to regulate the expression of different proteins within different compartments in a single neuronal cell type
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