Long-term memory involves the activity-induced synthesis of plasticity-related proteins in neurons to support long-lasting morphological and functional changes of synapses. This type of activity-driven gene expression is regulated at both transcriptional and translational levels (1–3
). Previous research has shown that proteins enriched at or near neuronal synapses accumulate in the nucleus in response to stimulation. These proteins include factors controlling gene expression, scaffold molecules, and the proteolyzed intracellular domains of synaptic receptors (4
). Transcription factors, such as cyclic AMP responsive element-binding protein (CREB)2, and translation regulators, such as cytoplasmic polyadenylation element-binding protein (CPEB)3 and CPEB4, move to the nucleus after activation of the N
-aspartate receptor (NMDAR) (6–9
), suggesting that the nucleocytoplasmic partition of these proteins by specific neuronal signaling determines their role in gene expression.
Synapse-to-nucleus communication (i.e. retrograde trafficking of protein molecules from synaptic areas to the nucleus) plays an important role in synaptic plasticity, long-term memory, circadian rhythms and neuronal survival (4
). However, the molecular machineries, including signal initiators and nucleocytoplasmic translocators, responsible for redistributing these synaptic proteins are less characterized. Although the small proteolyzed intracellular domains of receptors can passively diffuse into the nucleus, proteins with a molecular weight exceeding 40
kDa generally require the assistance of karyopherins for nuclear localization (11
). Importins α1, α2 and β1 of the nuclear import machinery are present at somatodendritic regions, and stimulation of neurons with NMDA increases the nuclear translocation of all three importins (12
). Importins α and β, in conjunction with nucleocytoplasm translocation regulators such as Ras-related nuclear protein (Ran) and Ran-binding protein (RanBP)1, are also present in the axoplasm of sciatic nerves (13
). After nerve injury, the expression of importin β and RanBP1 are upregulated (13
) that may help deliver transcription factors, such as signal transducer-activated transcription (Stat)3, to the nucleus (14
). The accumulation of nuclear CREB2 via importin α1/6-mediated translocation appears in neurons treated with NMDA to induce chemical long-term depression (c-LTD) (8
). NMDAR signaling also induces nuclear localization of Jacob by importin α1-facilitated translocation (7
). Thus, activity-regulated import pathways appear to play a critical role in transporting neuronal signals from distal dendrites and axons into the nucleus to control gene expression.
CPEB family proteins, including CPEBs1–4, regulate the translation of target-specific RNAs and reside predominantly in the cytoplasm. Recent research has identified that CPEBs 2 and 3, repress translation elongation through an interaction with eEF2 and stimulate translation in response to arsenite (16
) or NMDA (17
) in a polyadenylation-independent manner. Despite the cytoplasmic role of CPEBs in regulating translation, recent studies have shown that all CPEBs accumulate in the nucleus when chromosome region maintenance (CRM)1-dependent export is blocked by leptomycin B (LMB) in various cells, indicating that CPEBs are nucleocytoplasm-shuttling proteins (6
). Interestingly, CPEB3 and CPEB4 accumulate in the nucleus in NMDA-stimulated neurons (6
). Although CPEB4’s function in the nucleus is unclear, the nuclear form (i.e. export-defective mutant) of CPEB4 has a less protective effect in neurons deprived of oxygen and glucose (6
), a cellular model that mimics ischemia-induced neuronal death (19
). In contrast, CPEB3 can interact with Stat5b and inhibit Stat5b-dependent transcription in the nucleus (9
). Nonetheless, it remains elusive whether the source of elevated nuclear CPEB3 is contributed by a change in import or export in NMDA-stimulated neurons and whether specific karyopherins are involved to facilitate nuclear translocation of CPEB3. Although CPEB3, with a molecular weight of 76
kDa, likely needs an active import mechanism to transit across the nuclear pore complex, in silico
analysis does not reveal any canonical nuclear localization sequence (NLS) in CPEB3.
Using various CPEB3 mutants and small hairpin RNA (shRNA) knockdown screening, we have identified two cis-elements of CPEB3, the LENSL motif and RNA-recognition motif (RRM)1, control CPEB3’s nucleocytoplasmic distribution through interaction with CRM1 and importin 5 (IPO5), respectively. Time-lapse live imaging and biochemical experiments suggest that NMDA-increased RanBP1 expression presumably activates Ran’s GTPase. The conversion of GTP-bound (RanGTP) to GDP-bound Ran (RanGDP) facilitates IPO5’s binding to CPEB3 and increases the nuclear translocation of the Ran–IPO5–CPEB3 complex to elevate nuclear CPEB3 level.