However, the CPEB3 mRNA target actin 3 UTR (6) allowed for phase separation

However, the CPEB3 mRNA target actin 3 UTR (6) allowed for phase separation. the context of the aberrant phase separation that characterizes other RNA-binding proteins implicated in neurodegenerative diseases, such as FUS (fused in sarcoma) and TDP-43 (TAR DNA binding protein 43) in ALS (amyotrophic lateral sclerosis). CPEB and in yeast prions (3, 7, 8) exists in the N terminus of CPEB3, the proposed site that mediates oligomerization. Functionally, CPEB3 regulates the translation of mRNA targets, including AMPA-type glutamate receptor subunits GluA2 and GluA1, NMDA receptor subunit 1 (NR1), the cytoskeletal protein actin, and postsynaptic density protein 95 (PSD95) (4, 6, 9C11), all of which play major roles in synaptic plasticity (12C14). This regulation of translation of mRNA targets is connected to the structure of CPEB3. Soluble CPEB3 inhibits target mRNA translation while oligomeric, partially insoluble CPEB3 promotes the translation of target mRNA (4). As neurons are polarized structures, we presume that mRNAs involved in the maintenance of long-term memory will be under strict spatial control. Indeed, intracellular transport of mRNA and local translation play a key role in neuronal physiology. Translationally repressed mRNAs are transported to distant dendritic sites as part of ribonucleoprotein (RNP) particles. A new class of RNP particles, the dendritic P body-like structures (dlPbodies), has been recently described (15). These P body-like structures are present in the soma and dendrites of mammalian neurons. These particles show motorized movements along dendrites and relocalize to distant sites in response to synaptic activation (15). Dcp1a, a critical component of these structures, is stably associated with dlP bodies in unstimulated cells, but exchanges rapidly upon neuronal activation, concomitant with the loss of Ago2 from dlP bodies. Thus, dlP bodies may regulate local translation by storing repressed mRNPs in unstimulated cells, and releasing them Rabbit polyclonal to ANGPTL1 upon synaptic activation (15). In the present study, we examine the subcellular localization of CPEB3 and identify one possible mechanism that explains how CPEB3 mediates the translation of its mRNA targets, namely by residing in P bodies under resting conditions and translocating to polysomes upon synaptic activity. Results CPEB3 Is Localized to the Nucleus and Enters the Cytoplasm via a Nuclear Export Signal. Previous studies primarily examined the cellular distribution of the CPEB3 protein within the mouse brain tissue (9, 16). To gain further insight into CPEB3s function, we studied its subcellular localization both within human cells (HeLa), which express CPEB3 endogenously (16), and within cultured neuronal cells. The of Orb, a CPEB ortholog (20). We also performed immunofluorescence experiments to confirm our biochemical fractionation experiments and confirmed that CPEB3-GFP and CCR4 colocalize (and = 12 replicates per condition, ANOVA; 0.01, * 0.05, ** 0.01, TukeyCKramer post hoc analysis). Mock indicates transfection with empty vector. (= 6; ANOVA, with TukeyCKramer post hoc analysis; * 0.05, ** 0.01). (= 3); test reveals a significant difference (** 0.05). Chemical Long-Term Potentiation Promotes the Translocation of CPEB3 from P Bodies to Polysomes. CPEB3 is found both in the nucleus and in the cytoplasm. Within the cytoplasm, CPEB3 distributes with other proteins that participate in the regulation of translation. When analyzed by differential centrifugation of cell extract, basal CPEB3 in neurons resided in fractions containing the P-body component Dcp1 and the deadenylase complex subunit CCR4 ( 0.05, ** 0.01. One-way ANOVA and Tukeys multiple comparisons, with = 30, 3 technical replicates. ( 0.05. One-way ANOVA and Tukeys multiple comparisons, with = 30, 3 technical replicates. R(obs) represents the observed correlation of green and red signal, according to Fijis colocalization test [which utilizes Costess image randomization (100 iterations) and van Steensel and Fays image shift analysis (62)]. (and = 4 samples with technical triplicates ( 0.001). We used actin 3 UTR as the specific RNA target in these experiments, to which CPEB3 binds under both basal and stimulating conditions (6). Temperature, salt, and metal did not have an obvious influence on phase separation of CPEB3, nor did general HeLa mRNA (= 40; across 5 sample groups) or ginkgolic acid-treated (= 23; across 5 sample groups) samples. An asterisk represents statistical significance, = 0.0002, test. ( 0.05, ** 0.001, TukeyCKramer post hoc analysis). SUMOylation Drives CPEB3 Localization to P Bodies and Modulates the Ability of CPEB3 to Repress Translation of Its Target mRNAs. Thus far, our data suggest that in vivo CPEB3 is located in phase-separated WAY-600 RNA granules, such as P bodies. In vitro, we observed that CPEB3 phase separated when SUMOylated WAY-600 and WAY-600 bound to a specific target mRNA. To link the in vivo and in vitro findings, we utilized a CPEB3-GFP construct and ginkgolic acid treatment, which inhibits SUMOylation (35). When SUMOylation is inhibited, transfected CPEB3-GFP colocalizes less with the P-body marker Dcp1 (Fig. 5and WAY-600 human CPEB4 regulates phase separation of the protein (44). There are examples of SUMOylation in the nucleus promoting.