A strategy to capture and characterize the synaptic transcriptome

Abstract

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.long-term memory storage | RNA transport | local protein synthesis | neurogenomics | cystoskeleton D espite decades of research on the importance of local translation in learning-related synaptic plasticity and long-term memory storage, we know relatively little about the identity of the "synaptic transcriptome," the various components of the total RNA population that is transported from the cell body, and how this transcriptome becomes localized to and translated at specific synapses (1-12). To develop a general strategy for isolating and characterizing all of the RNAs transported from the cell body to the synapse, we have focused on the RNA transport complexes that interact with the molecular motor kinesin that mediate transport of gene products from the cell body to synapses (13,14).Our approach offers four distinct advantages over previously described methods: (i) it allows the identification of RNAs based on their association with transport machinery that is destined for synapses; (ii) it reflects dynamically regulated RNAs; (iii) it allows for identification of the targeted RNAs; and (iv) it can be used in different regions of the central nervous system (CNS), thereby facilitating genomic characterization of synaptic transcriptome of the entire CNS or region of the CNS. These advantages should aid the study of stoichiometric changes in localized RNAs and their role of local translation, not only in memory storage, but also in a variety of other physiological conditions, such as development. Using this strategy, we have succeeded in obtaining a comprehensive collection of RNAs targeted to Aplysia CNS synapses. We further show that myosin heavy chain mRNA, a cargo of kinesin, is localized to sensory neuron processes and is required specifically for the induction of long-term facilitation (LTF) at sensory and motor neuron synapses. Results Strategy for Identifying Synaptically Targeted RNAs: Isolation andCharacterization of RNA Transport Complexes. We assumed that successful isolation of the RNA-protein complexes associated with the kinesin transport machinery would help identify the full repertoire of RNAs that are actively transported to synapses. Because the CNS contains both neuronal and nonneuronal cells, this approach will also yield RNAs found in the kinesin complex in nonneuronal cells, such as glia. We first optimized conditions for isolating RNA transport complexes from the CNS of Aplysia based on a previous...

RNA transport and long-term memory storage

“…However, recent studies of RNA localization in mice, 5 Aplysia, 52 and proteomics analysis of signaling complexes at the synapse, [76][77][78] suggest an unprecedented molecular complexity of the synapse. Translational control by miRNAs at the synapse adds another layer to this complexity.…”

“…40 Knockdown experiments of ApMHC, actin-dependent molecular motor, and mRNA cargo of ApKHC1, also showed similar results on initiation and persistence of LTF. 52 Together, these studies suggest that during persistence of memory, synapses become autonomous with respect to transport from the cell body, and RNAs are stored at the synapses for later use.…”

“…52 This unbiased analysis of RNAs in the ApKHC1 complex also identified several non-coding RNAs, such as antisense RNAs, 52 whose specific function at the synapse is yet to be determined.…”

New approach to capture and characterize synaptic proteome

“…The composition of the synaptic proteome is determined by proteins transported from the cell body and local synthesis of proteins at the synapses. Several studies have shown the identification and localization of RNAs at the synapse (9)(10)(11)(12). Comprehensive knowledge of the proteins that are actively targeted to synapses will help elucidate the dynamics of signal transduction at the synapse, as well as the molecules that are critical for the remodeling of synapses and formation of new synapses that accompany long-term memory storage.…”

“…To identify actively transported proteins, we focused on the protein complexes associated with kinesins, molecular motor proteins that move from the cell body to distal neuronal processes and mediate the transport of various gene products (9,13). We considered that proteins associated with kinesins include both synaptic proteins and proteins localized to dendrites and axons.…”