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...

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Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.DOI: http://dx.doi.org/10.7554/eLife.12821.001

Programmed cell death is an essential aspect of animal development. Mutations in vertebrate genes that mediate apoptosis only mildly perturb development, suggesting that other cell death modes likely have important roles. Linker cell-type death (LCD) is a morphologically conserved cell death form operating during the development of Caenorhabditis elegans and vertebrates. We recently described a molecular network governing LCD in C. elegans, delineating a key role for the transcription factor heat-shock factor 1 (HSF-1). Although HSF-1 functions to protect cells from stress in many settings by inducing expression of protein folding chaperones, it promotes LCD by inducing expression of the conserved E2 ubiquitin-conjugating enzyme LET-70/UBE2D2, which is not induced by stress. Following whole-genome RNA interference and candidate gene screens, we identified and characterized four conserved regulators required for LCD. Here we show that two of these, NOB-1/Hox and EOR-1/PLZF, act upstream of HSF-1, in the context of Wnt signaling. A third protein, NHR-67/TLX/NR2E1, also functions upstream of HSF-1, and has a separate activity that prevents precocious expression of HSF-1 transcriptional targets. We demonstrate that the SET-16/mixed lineage leukemia 3/4 (MLL3/4) chromatin regulation complex functions at the same step or downstream of HSF-1 to control LET-70/UBE2D2 expression. Our results identify conserved proteins governing LCD, and demonstrate that transcriptional regulators influence this process at multiple levels.

The nematode Caenorhabditis. elegans has served as a fruitful setting for cell death research for over three decades. A conserved pathway of four genes, egl-1/BH3-only, ced-9/Bcl-2, ced-4/Apaf-1, and ced-3/caspase, coordinates most developmental cell deaths in C. elegans. However, other cell death forms, programmed and pathological, have also been described in this animal. Some of these share morphological and/or molecular similarities with the canonical apoptotic pathway, while others do not. Indeed, recent studies suggest the existence of an entirely novel mode of programmed developmental cell destruction that may also be conserved beyond nematodes. Here we review evidence for these noncanonical pathways. We propose that different cell death modalities can function as backup mechanisms for apoptosis, or as tailor-made programs that allow specific dying cells to be efficiently cleared from the animal.