3′UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons

Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. Here, we show that the 3′UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3′UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise. We found that the phenotypic effects of the orb2 3′UTR deletion were rescued by introducing the 3′UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3′UTR deletion were not rescued by the 3′UTR from one of the Drosophila α-tubulin genes. Our results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3′UTR.


Introduction
Proper distribution of mRNAs and proteins within the cell plays a critical role in embryogenesis, cell differentiation, and the functioning of specialized cell types [1][2][3][4]. As is known, mRNA localization coupled with on-site translation is one of the common strategies used to localize proteins to appropriate cell compartments [5][6][7]. Neurons, which have long outgrowths, termed axons, and much shorter outgrowths, dendrites, are a context in which mRNA localization and on-site translation are particularly important. Specific mRNAs and proteins must be localized in the distal part of neuronal outgrowths (neuritis) [8,9] to ensure synaptic transduction of nerve impulses between neurons [10].
Cytoplasmic polyadenylation element (CPE) binding proteins (CPEBs) are a family of proteins that are known to play a key role in on-site translational regulation [11]. Depending on the context and the mRNA, CPEBs can either repress or activate translation through a polyA-dependent mechanism [12,13]. In addition to regulating translation, CPEBs can mediate the intracellular transport of mRNAs in neurons according to several studies [14][15][16].

Generation of Plasmid Constructs for Drosophila Transgenesis: Fly Strains
Full-length 3 UTR sequences from the alpha-tubulin (αTub84B) and orb genes were amplified from genomic DNA with primers containing BamHI and BglII restriction sites. The sequences of primers and the 3 UTRs are given in the Supplementary Materials. Next, the amplified fragments were cloned in a pOT2-attB-containing vector. The resulting plasmids were checked by sequencing.
In the starting orb2 3 UTR deletion, orb2 R , an attP site for recombination was substituted for the deleted 3 UTR sequences [35]. To generate the replacements, the orb2 R fly strain was crossed with a phiC31-expressing strain (34772, DBSC, USA, genotype: y (1) w(*) P[y[+t7.7] = nos-phiC311]X; L (1)/CyO; TM2/TM6B, Tb (1)). Embryos obtained from the crosses were injected with a plasmid containing attB and the full-length sequences of the αTub84B and orb 3 UTRs. Flies with recombination events were selected by dsRed expression in the eyes; the dsRed marker was removed via Cre recombinase-mediated excision. Five independent flies with recombination events were used to generate homozygote stocks; the integrated fragments were sequenced. The resulting strains were designated as orb2 tub and orb2 orb . The Drosophila fly strain 51324 (genotype: w (1118); PBacVK00027) from the Drosophila Bloomington Stock Center (DBSC, Bloomington, IN, USA) was additionally used in the study as a wild-type (WT) control to measure the mRNA and protein levels. It was shown previously that the 51,324 fly strain has the same ability to form memory as the standard CantonS strain, which is usually used in behavioral assays. The CantonS fly strain was used as a WT control in behavioral experiments.

Antibodies
The 4G8 (1:50 dilution) antibody against the Orb2 protein was used in Western blotting. The antibody was deposited in the Developmental Studies Hybridoma Bank (DSHB, Iowa, IA, USA) by P. Schedl. The DCSP-1 (ab49) antibody against the Cysteine-string protein (CSP) was deposited in the DSHB by E. Buchner and A. Hofbauer and was used in a 1:500 dilution. The 3C11 antibody against Synapsin was deposited in the DSHB by B.R.E. Klagge and colleagues and used at a 1:200 dilution. An anti-polychaetoid (PYD1) antibody was deposited in the DSHB by A.S. Fanning and used at a 1:500 dilution. An HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, UK, catalog number 115-035-174) was used as a secondary antibody in Western blotting at 1:2000.

Behavioral Assay
A courtship-suppression paradigm was used to assess memory formation [32,36]. Flies were raised on standard Drosophila yeast-raisin medium at 25 • C and 60-70% humidity with a 12 h light-dark cycle. Males of a studied strain were collected without anesthesia 0-10 h after eclosion and kept individually in food vials until the tests. The training was performed using 5-day-old CS females fertilized 1 day before. Testing was performed at the age of 5 days. For training, naive males having no courtship experience were placed in a chamber with a fertilized female for 30 min for further assessment of either STM (short-term memory, immediately after training) or MTM (middle-term memory, 180 min. after training). For LTM formation, a male and a female were kept together for 5 h and assessed 2 days after training, using newly fertilized 5-day-old CS females. Naive males were used as a control. For each point, 20 males were tested. The time spent in courtship (orientation, following, wing vibration, licking, and attempted copulation) was recorded for 300 s. The courtship index (percentage of time spent in courtship) was calculated for each male. A courtship index (CI) was calculated as a ratio of courtship time to the entire observation time [36]. A learning index (LI) was calculated as LI = [(CIn − CIexp)/CIn] × 100%, where CIn is the average CI of two independent samples of males without courtship experience, and CIexp is the average CI of two independent samples of males after training. Statistical comparisons of behavioral data were conducted by a randomization test, by directly calculating the probability of rejection of the null hypothesis αR. A sampled randomization test with 10,000 permutations was used. The null hypothesis was rejected at αR < 0.05. A two-sided test was used to compare CIs, and a one-sided test for the comparison of LIs. In the latter case, no SEMs were required [36].

Fluorescent In Situ Hybridization (FISH)
To perform in situ hybridization, at least 20 brains were dissected from 4-to 5-day-old flies for each genotype used in the study. The brains were washed twice with PBS supplemented with 0.05% Tween-20 (PBSt) for 10 min and fixed with 3.5% paraformaldehyde for 20 min at room temperature. The brains were treated with 100% methanol at -20 • C for 5 min, and then methanol was replaced with PBS through a series of washes with descending vol/vol methanol/PBS solutions (70/30, 50/50, 30/70). Each rinse was combined with rotation and lasted 10 min (room temperature). After the final wash, the brains were washed with PBSt twice for 5 min and incubated in a prewarmed wash buffer (4× SSC, 35% formamide, 0.1% Tween-20) at 37 • C for 15 min. After incubation, the samples were placed in a hybridization buffer (Stellaris RNA FISH Hybridization Buffer, LGC Biosearch Technologies, Middlesex, UK, cat. No SMF-HB1-10) supplemented with FISH probes at a 1:10 dilution and incubated at 37 • C with continuous shaking. The next day, the samples were washed with a wash buffer at 37 • C for 4 h and with PBSt at room temperature for 2 h. The samples were mounted on a slide glass in the VectaShield mounting medium with DAPI (Vector Labs, Newark, CA, USA) and examined by microscopy. The FISH probes were manufactured by LGC Biosearch Technologies. A probe set consisted of twenty 20-nt probes complementary to part of the last common exon of the orb2 gene. Each 20-nt probe was conjugated with Quasar 670 at the 3 end. The sequences of all probes are presented in the Supplementary Materials. When performing quantitative PCR, each sample was used in technical triplicate. At least five independent biological replicas were used in every experiment. To assess the relative amount of the orb2 mRNA in total fly head extracts, gapdh2 was used as a reference gene to calculate 2 −∆∆Ct . The 28S RNA was used as a reference when 2 −∆∆Ct was calculated to compare the distribution of the orb2 mRNA and its potential mRNA targets (act5C, csp, and pyd) between the cell body and synaptic fractions of neurons. Sequences of primers used in the study are presented in the Supplementary Materials.

Crude Synaptosome Preparation
To study the protein distribution in nerve cells, 50 fly heads of each genotype were homogenized in 50 µL of 0.32 M sucrose buffer (5 mM HEPES, pH 7.4, 145 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose) supplemented with a protease inhibitor cocktail. Crude synaptosome preparations were obtained essentially as described in [37] with an extra centrifugation step added after collecting the first pellet. To study the RNA distribution, we collected at least 400 flies of each genotype. The flies were frozen in liquid nitrogen, vortexed, and sifted through two 800 and 380 µm sieves to separate heads from bodies. The 0.32 M sucrose homogenization buffer was additionally supplemented with a ribonuclease inhibitor (Ribolock, Thermo Fisher Scientific, Waltham, MA, USA, catalog number EO0382).

Semiquantitative Western Blot Analysis
To assess the distribution of the proteins of interest in Drosophila nervous cells, we used the strategy described in [32]. Twenty fly heads of each genotype were taken for analysis.

Confocal Microscopy
Stained samples of fly brains were scanned and imaged under a Leica STELLARIS 5 (Wetzlar, Germany) confocal microscope. We used a 40× oil objective with a numerical aperture of 1.3. Images with a frame size of 2048 × 2048 pixels and a z resolution of 1 µm were taken at a scan speed of 400. At least four brains were imaged for each genotype, and selected images were processed using LazX software (Leica Application Suite X, version 3.7.25997.6., Leica Microsystems, Wetzlar, Germany). Average intensity values in the marked brain regions were determined using Fiji 1.53t software.

Quantification and Statistical Analysis
Experimental data were statistically analyzed with GraphPad Prism 8.0.2. software. Graphs presented in the figures were plotted using the same software. Columns display the mean values; error bars display the standard deviation. Two-tailed Student's t-test was used to compare the relative mRNA and protein concentrations between flies with different genotypes. Asterisks in the graphs indicate p-value < 0.05; ns, nonsignificant differences.

Generation of Fly Strains with Replacements of Deleted Part of orb2 3 UTR
In previous studies, we generated a 4.5 kb deletion of sequences encoding two of the larger orb2 3 UTRs [35]. As indicated in Figure 1, the largest orb2 3 UTR has 37 CPElike elements, while the orb2 R deletions have only four putative CPE elements, which are located closer to the stop codon for Orb2B (hereinafter referred to as Orb2) protein. The deletion does not remove sequences from the 3 UTR sequences of the mRNA encoding the Orb2A isoform, nor does it remove sequences from the shortest orb2B mRNA, orb2-RB (see Figure 1). Analysis of the starting orb2 R deletion showed that Orb2 protein levels in the synaptic zone of neurons were reduced, while the levels in neuronal cell bodies were equivalent to those in WT. We also found that the formation of long-term memory (LTM) in the orb2 R mutant was significantly impaired [32].

Introduction of Different Sequences in orb2 R Allele Does Not Affect Short-Term and Mid-Term Memory Formation but Affects Long-Term Memory
The CIs of orb2 tub and orb2 orb adult males were assessed to evaluate their ability to form short-term (STM) and mid-term memory (MTM) ( Figure S1). Figure 1B summarizes the calculated LIs. As expected, the CIs of orb2 tub flies were similar to the CIs of WT flies. The same picture was observed in orb2 orb flies-i.e., their STM and MTM formation was normal ( Figure 1B). Thus, we found that different sequences of the 3′UTR of the orb2 mRNA did To provide further evidence that the proper localization/regulation of orb2 mRNA is critical for LTM formation, we replaced the deletion with two different UTR sequences, the αTub84B tubulin gene 3 UTR, which contains a single consensus CPE sequence, and the 3 UTR that belongs to the orb, another CPEB gene, and contains eight CPEs [34,38]. The full sequences are presented in Materials and Methods. We chose these two test UTRs because in the orb2 R deletion, the remaining UTR sequences (see Figure 1A) had only four CPE-like elements and we supposed that among other things, the number of CPE sequences likely played a critical role in the localization of the orb2 mRNA and/or its efficient translation. In addition, other regulatory motifs were found in the 3 UTR of the orb2 mRNA, namely, the bird-box motif, Bruno motifs, and Musashi binding elements (MSI) [39,40]. All of them were also present in the orb 3 UTR. A Pumilio binding motif (PBE) is additionally found in the 3 UTR of the orb mRNA [41].
After phiC31-mediated integration of the UTR sequences in the attP site, the dsRed marker was removed. The final fly strains used in further experiments were designated as orb2 tub and orb2 orb ( Figure 1B).

Introduction of Different Sequences in orb2 R Allele Does Not Affect Short-Term and Mid-Term Memory Formation but Affects Long-Term Memory
The CIs of orb2 tub and orb2 orb adult males were assessed to evaluate their ability to form short-term (STM) and mid-term memory (MTM) ( Figure S1). Figure 1B summarizes the calculated LIs. As expected, the CIs of orb2 tub flies were similar to the CIs of WT flies. The same picture was observed in orb2 orb flies-i.e., their STM and MTM formation was normal ( Figure 1B). Thus, we found that different sequences of the 3 UTR of the orb2 mRNA did not affect the STM and MTM formation, as has been shown for the initial orb2 R fly strain.
Next, we studied how different 3 UTR sequences affect the LTM formation when added to the orb2-coding region. The αTub84B 3 UTR did not restore the LTM formation, and orb2 tub flies showed the same CIs as naive males 2 days after training. In contrast, orb2 orb flies showed a significant reduction in CI just after training and retained this reduction 2 days after training. Thus, a replacement of the 3 UTR sequence deleted from the orb2 gene with the orb 3 UTR sequence leads to complete recovery of the LTM formation.

Different 3 UTR Sequences Used to Replace Deletion of orb2 3 UTR Do Not Influence mRNA Level but Affect Protein Level of Orb2 in Total Fly Head Extracts
To better understand why LTM formation in orb2 tub and orb2 orb males is different, we assessed how insertions into the orb2 R allele affect the orb2 mRNA level. The relative orb2 mRNA level mRNA was calculated by the ∆∆Ct method with gapdh2 used as a reference gene. The results are presented in Figure 2A. We found that the orb2 mRNA level did not differ from the WT level in both the orb2 tub and orb2 orb fly strains. The same result was observed in the orb2 R strain [32]. Thus, the insertion of an additional sequence in place of the deleted part of the orb2 gene does not change the relative production or the stability of the orb2 mRNA. Next, the Orb2 protein level was assessed in total fly head extracts of the orb2 tub and orb2 orb fly strains. Chemiluminescent signals from staining with antibodies against the Orb2 protein were normalized to the total protein in the sample. We found that orb2 orb flies had the same Orb2 protein level as WT flies. In contrast, orb2 tub flies showed a 34% decrease in Orb2 protein level as compared with WT flies ( Figure 3B). The results demonstrate that the orb 3 UTR sequence restored the total Orb2 protein amount in the fly brain, while the tubulin gene 3 UTR did not elevate the Orb2 protein amount to the WT level. The orb2 tub and initial orb2 R strains showed a similar level of decreasing of Orb2 protein amount.

3 UTR of orb2 Gene Is Essential for Intracellular Transport of orb2 mRNA
The next question to study was how the sequences introduced into the orb2 3 UTR influence the orb2 mRNA intracellular distribution. We applied the previously used biochemical approach to separate the soma and synaptic regions [32]. The results are presented in Figure 3A. We did not find any difference in the orb2 mRNA level from the WT in the soma fraction of neurons in both the orb2 tub and the orb2 orb fly strains. The synaptic fraction of the orb2 orb strain was similar to the WT in the amount of the orb2 mRNA. In contrast, the orb2 tub synaptic fraction displayed a nearly threefold reduction in orb2 mRNA compared with the WT. We checked the distribution of the orb2 mRNA in the initial orb2 R fly train. The amount of the orb2 mRNA in the soma of neurons was found to be the same as in the WT, whereas a decrease in the orb2 mRNA level was observed in the synaptic fraction, similar to the orb2 tub strain. Thus, we can conclude that the orb2 3 UTR sequence is essential for localizing the orb2 mRNA to the synaptic compartment of the nerve cell.
, 12, x FOR PEER REVIEW 7 of 17 mRNA level mRNA was calculated by the ΔΔCt method with gapdh2 used as a reference gene. The results are presented in Figure 2A. We found that the orb2 mRNA level did not differ from the WT level in both the orb2 tub and orb2 orb fly strains. The same result was observed in the orb2 R strain [32]. Thus, the insertion of an additional sequence in place of the deleted part of the orb2 gene does not change the relative production or the stability of the orb2 mRNA. Columns show the mean mRNA levels detected in orb2 tub and orb2 orb (t-test, n = 6, SD is shown). (B) Relative levels of the Orb2 protein in total head extracts from orb2 tub and orb2 orb flies. A red dashed line presents the Orb2 protein level in WT flies, which was used for normalization. The mean Orb2 protein level was found to decrease in orb2 tub flies (n = 6, SD is shown, asterisk shows p < 0.05), while orb2 orb flies had the same Orb2 protein level as WT flies. (C) Western blot analysis of head lysates from orb2 tub and orb2 orb flies with antibodies against the Orb2 protein (top). Total protein staining (bottom) was used for normalization in a twofold serial dilution. Next, the Orb2 protein level was assessed in total fly head extracts of the orb2 tub and orb2 orb fly strains. Chemiluminescent signals from staining with antibodies against the Orb2 protein were normalized to the total protein in the sample. We found that orb2 orb flies had the same Orb2 protein level as WT flies. In contrast, orb2 tub flies showed a 34% decrease in Orb2 protein level as compared with WT flies ( Figure 3B). The results demonstrate that the orb 3′UTR sequence restored the total Orb2 protein amount in the fly brain, while the tubulin gene 3′UTR did not elevate the Orb2 protein amount to the WT level. The orb2 tub and initial orb2 R strains showed a similar level of decreasing of Orb2 protein amount. presents the orb2 mRNA level in WT flies, which was used for normalization. Columns show the mean mRNA levels detected in orb2 tub and orb2 orb (t-test, n = 6, SD is shown). (B) Relative levels of the Orb2 protein in total head extracts from orb2 tub and orb2 orb flies. A red dashed line presents the Orb2 protein level in WT flies, which was used for normalization. The mean Orb2 protein level was found to decrease in orb2 tub flies (n = 6, SD is shown, asterisk shows p < 0.05), while orb2 orb flies had the same Orb2 protein level as WT flies. (C) Western blot analysis of head lysates from orb2 tub and orb2 orb flies with antibodies against the Orb2 protein (top). Total protein staining (bottom) was used for normalization in a twofold serial dilution.

3 UTR of orb2 Gene Is Essential for Orb2 Protein Distribution
To analyze the Orb2 protein distribution in neurons of the orb2 tub and orb2 orb fly strains, we prepared the fractions of cell bodies and synaptosomes. We found that the Orb2 protein amount in the cell bodies of the strains did not differ from that in WT flies. In the synaptosome fraction, the Orb2 protein amount in orb2 orb flies did not differ from that in WT flies, whereas orb2 tub flies showed a more than twofold reduction in the Orb2 amount ( Figure 3A). Thus, the distribution of the Orb2 protein among the fractions in orb2 tub flies was similar to that reported previously for orb2 R flies [32]. The results have led us to the conclusion that the 3 UTR of the orb2 gene is essential for the proper localization of the orb2 mRNA and protein to synapses.

3′UTR of orb2 Gene Is Essential for Intracellular Transport of orb2 mRNA
The next question to study was how the sequences introduced into the orb2 influence the orb2 mRNA intracellular distribution. We applied the previously us chemical approach to separate the soma and synaptic regions [32]. The results a sented in Figure 3A. We did not find any difference in the orb2 mRNA level from t in the soma fraction of neurons in both the orb2 tub and the orb2 orb fly strains. The sy fraction of the orb2 orb strain was similar to the WT in the amount of the orb2 mR contrast, the orb2 tub synaptic fraction displayed a nearly threefold reduction in orb2 compared with the WT. We checked the distribution of the orb2 mRNA in the initia fly train. The amount of the orb2 mRNA in the soma of neurons was found to be th as in the WT, whereas a decrease in the orb2 mRNA level was observed in the sy fraction, similar to the orb2 tub strain. Thus, we can conclude that the orb2 3′UTR se is essential for localizing the orb2 mRNA to the synaptic compartment of the nerve

Sequence in 3 UTR of orb2 Is Important for mRNA Localization to Neuron Outgrowths
To more precisely study the mRNA distribution in the fly brain for different orb2 alleles, in situ hybridization with Stellaris probes was performed using whole-mount brain samples. The orb2 mRNA was abundantly present in neuropile regions in WT flies ( Figures 4A, S2 and S3). We also found the orb2 mRNA in the soma of neurons, as has been shown before [23]. When studying orb2 R fly brains, we found that staining was decreased in neuropile regions and that the signal from the orb2 probe was distributed evenly in different parts of the brain as compared with the WT (Figures 4A, S2 and S3). We quantified the signal intensities of the orb2 mRNA in the neuropile and soma regions and then calculated their ratio. The relative intensity of the orb2 mRNA in the neuropile regions is significantly reduced in orb2 R mutants ( Figure 4B).  A hybridization pattern similar to the orb2 R pattern was observed in orb2 tub samples. Regions enriched in axons were poorly stained with the orb2 probe ( Figures 4A, S2 and S3). In contrast, a recovery of the orb2 mRNA distribution observed in WT brains was detected in orb2 orb brains. This is confirmed by a quantitative assessment of staining intensity ( Figure 4B).
An interesting feature of the orb2 mRNA distribution was additionally observed in the neuron soma. A perinuclear ring with several bright speckles per cell was formed in the region of the calyx (dendritic input for Kenyon cells) by the orb2 mRNA ( Figures S4 and S5). The speckles were clearly observed in WT flies. Sites of a high local concentration of the orb2 mRNA in the soma may be assumed to act as sites of RNP assembly. The same picture was found in orb2 orb brains. In orb2 R and orb2 tub flies, the perinuclear ring was preserved, but the speckles were hardly distinct from other orb2 mRNA fractions localized in the soma. In addition, the orb2 mRNA was distributed more evenly between the cell soma and the calyx in orb2 R and orb2 tub brains and the background staining was stronger compared with the WT.
The changes in orb2 mRNA distribution observed in orb2 R and orb2 tub could be attributed to changes in brain structures. To check this assumption, brain samples were stained with antibodies against Synapsin, which is a marker of neuropile regions. The results are presented in Figure 5. We did not find any obvious change in brain organization of the orb2 R , orb2 tub , and orb2 orb strains compared with the WT. An interesting feature of the orb2 mRNA distribution was additionally observed in the neuron soma. A perinuclear ring with several bright speckles per cell was formed in the region of the calyx (dendritic input for Kenyon cells) by the orb2 mRNA ( Figures S4  and S5). The speckles were clearly observed in WT flies. Sites of a high local concentration of the orb2 mRNA in the soma may be assumed to act as sites of RNP assembly. The same picture was found in orb2 orb brains. In orb2 R and orb2 tub flies, the perinuclear ring was preserved, but the speckles were hardly distinct from other orb2 mRNA fractions localized in the soma. In addition, the orb2 mRNA was distributed more evenly between the cell soma and the calyx in orb2 R and orb2 tub brains and the background staining was stronger compared with the WT.
The changes in orb2 mRNA distribution observed in orb2 R and orb2 tub could be attributed to changes in brain structures. To check this assumption, brain samples were stained with antibodies against Synapsin, which is a marker of neuropile regions. The results are presented in Figure 5. We did not find any obvious change in brain organization of the orb2 R , orb2 tub , and orb2 orb strains compared with the WT. Figure 5. The brain structure of the studied mutants. Staining with antibodies against synapsin. The brain structure in the strains orb2 R , orb2 tub , and orb2 orb is indistinguishable from that in WT brains. Scale bar, 50 µm.

Distribution of mRNAs and Proteins of Potential Orb2 Targets in Nerve Cells
We have previously found that the Csp and Pyd proteins, whose mRNAs are potential targets of the Orb2 protein, elevate their representation in synapses of orb2 R flies. csp encodes a chaperone associated with synaptic vesicles, and pyd encodes a protein involved in cell adhesion, which is important for mushroom body formation. The mRNAs encoding the proteins are thought to be regulatory targets of Orb2. One explanation for the increased amount of Csp and Pyd proteins in the synaptic fraction is that Csp and Pyd mRNAs are transported more efficiently and/or stabilized in the orb2 R deletion mutant, resulting in increased total mRNA levels encoding these two proteins. Alternatively, both mRNAs may be subject to translational repression by Orb2. In this case, a decrease in the level of Orb2 in the synapse would result in an increase in the translation of csp and pyd mRNA.
To explore these possibilities, we used qPCR to measure the amount of csp and pyd mRNA in soma and synapse fractions in the head obtained from orb2 R , orb2 tub, and orb2 orb flies. As shown in Figure 6A, the relative level and distribution in the soma and synaptic fractions were the same as in WT.
Next, we examined the distribution of Csp and Pyd proteins in the soma and synaptic fractions. In the orb2 orb sample, the levels of the proteins were the same as in the WT flies. However, in orb2 tub samples, the levels of the proteins were elevated in the synaptic fraction compared with the WT (Figure 6B). Similar results have been obtained previously for the orb2 R deletion [32]. Figure 5. The brain structure of the studied mutants. Staining with antibodies against synapsin. The brain structure in the strains orb2 R , orb2 tub , and orb2 orb is indistinguishable from that in WT brains. Scale bar, 50 µm.

Distribution of mRNAs and Proteins of Potential Orb2 Targets in Nerve Cells
We have previously found that the Csp and Pyd proteins, whose mRNAs are potential targets of the Orb2 protein, elevate their representation in synapses of orb2 R flies. csp encodes a chaperone associated with synaptic vesicles, and pyd encodes a protein involved in cell adhesion, which is important for mushroom body formation. The mRNAs encoding the proteins are thought to be regulatory targets of Orb2. One explanation for the increased amount of Csp and Pyd proteins in the synaptic fraction is that Csp and Pyd mRNAs are transported more efficiently and/or stabilized in the orb2 R deletion mutant, resulting in increased total mRNA levels encoding these two proteins. Alternatively, both mRNAs may be subject to translational repression by Orb2. In this case, a decrease in the level of Orb2 in the synapse would result in an increase in the translation of csp and pyd mRNA.
To explore these possibilities, we used qPCR to measure the amount of csp and pyd mRNA in soma and synapse fractions in the head obtained from orb2 R , orb2 tub, and orb2 orb flies. As shown in Figure 6A, the relative level and distribution in the soma and synaptic fractions were the same as in WT.
Changes in distribution were also explored for the actin5C mRNA in the fly strains because the mRNA has been shown to provide a potential target for the Orb2 protein [42,43]. We found that its amount significantly decreased (almost three times as compared with WT flies) in the synaptic fraction in the orb2 tub strain and was almost the same as in WT flies in the orb2 orb fly strain ( Figure 6A).

Discussion
One of the most important discoveries in neurosciences is that the synapse, rather than a single neuron, is an elementary unit for memory formation [44]. Presynaptic and postsynaptic termini contain unique sets of molecules for the transmission and reception of nerve impulses. A mechanism to target key proteins to the pre-and postsynaptic termini relies on mRNA transport followed by local translation [7,9]. Perhaps for this reason, many neuronal mRNAs have extended 3′UTRs compared with mRNAs encoding the same proteins in non-neuronal tissues [45].
Previously, we have reported the isolation and characterization of a large deletion (4.5 kb) that removes most of the sequence for two large orb2 3′UTRs [32]. The longest of the orb2 3′UTRs (orb2-RH) has 37 canonical and noncanonical CPE motifs, and all but 4 of these are preserved in flies with our orb2 R deletion. The deletion does not appear to impact the translation of the orb2 mRNAs in the neuronal cell body, and the levels of the Orb2 protein in this cell compartment are similar to that in the WT. On the other hand, the deletion reduces the level of the orb2 mRNA in synaptic fractions by more than half. A concomitant reduction in Orb2 protein in the synaptic fraction accompanies this reduction in mRNA.
In the studies reported here, we used a 3′UTR replacement strategy to rescue the orb2 R 3′UTR deletion. We tested the 3′UTRs derived from two different fly genes, the αTub84B tubulin gene and the orb gene for the other Drosophila CPEB protein. The former has a single CPE, while the latter has eight canonical CPEs. The αTub84B 3′UTR does not rescue Nerve cell fractions were obtained from WT, orb2 tub , and orb2 orb brain samples.
Next, we examined the distribution of Csp and Pyd proteins in the soma and synaptic fractions. In the orb2 orb sample, the levels of the proteins were the same as in the WT flies. However, in orb2 tub samples, the levels of the proteins were elevated in the synaptic fraction compared with the WT ( Figure 6B). Similar results have been obtained previously for the orb2 R deletion [32].
Changes in distribution were also explored for the actin5C mRNA in the fly strains because the mRNA has been shown to provide a potential target for the Orb2 protein [42,43]. We found that its amount significantly decreased (almost three times as compared with WT flies) in the synaptic fraction in the orb2 tub strain and was almost the same as in WT flies in the orb2 orb fly strain ( Figure 6A).

Discussion
One of the most important discoveries in neurosciences is that the synapse, rather than a single neuron, is an elementary unit for memory formation [44]. Presynaptic and postsynaptic termini contain unique sets of molecules for the transmission and reception of nerve impulses. A mechanism to target key proteins to the pre-and postsynaptic termini relies on mRNA transport followed by local translation [7,9]. Perhaps for this reason, many neuronal mRNAs have extended 3 UTRs compared with mRNAs encoding the same proteins in non-neuronal tissues [45].
Previously, we have reported the isolation and characterization of a large deletion (4.5 kb) that removes most of the sequence for two large orb2 3 UTRs [32]. The longest of the orb2 3 UTRs (orb2-RH) has 37 canonical and noncanonical CPE motifs, and all but 4 of these are preserved in flies with our orb2 R deletion. The deletion does not appear to impact the translation of the orb2 mRNAs in the neuronal cell body, and the levels of the Orb2 protein in this cell compartment are similar to that in the WT. On the other hand, the deletion reduces the level of the orb2 mRNA in synaptic fractions by more than half. A concomitant reduction in Orb2 protein in the synaptic fraction accompanies this reduction in mRNA.
In the studies reported here, we used a 3 UTR replacement strategy to rescue the orb2 R 3 UTR deletion. We tested the 3 UTRs derived from two different fly genes, the αTub84B tubulin gene and the orb gene for the other Drosophila CPEB protein. The former has a single CPE, while the latter has eight canonical CPEs. The αTub84B 3 UTR does not rescue the localization of the orb2 mRNA to the synaptic fractions. Moreover, like in the starting orb2 R deletion mutant, Orb2 protein levels in the synaptic fraction are reduced, while the protein amount in the neuronal cell body is similar to the WT. Consistent with the idea that localization of the orb2 mRNA to synaptic fractions is important for the orb2 function, orb2 tub flies exhibit phenotypes similar to those observed for orb2 R . These include elevated levels of the Csp and Pyd proteins in the synaptic fraction and pronounced deficits in LTM formation. The suggestion is supported by our analysis of the orb 3 UTR replacement of orb2 orb . This orb 3 UTR replacement appears to fully rescue the phenotype of the orb2 3 UTR deletion. Both orb2 mRNA and protein levels in the synaptic fraction are equivalent to the WT, as are the levels of the Csp and Pyd proteins. In addition, there is no obvious deficit in LTM formation.
Orb is known to be essential for LTM formation [33]. It is also known that the orb 3 UTR is essential for the proper localization of the Orb protein in the oocyte [38] through an autoregulatory loop. Additionally, the orb 3 UTR contains the BRE, MSI, and PBE motifs, as well as binding sites for the Pumilio protein, which could be essential for proper translation activation [39][40][41]. Fragments of similarity between the orb and orb2 3 UTRs are not long enough, but all of them contain multiple CPE sequences. Based on this observation, we assume that the number of CPE sequences in the orb2 3 UTR together with other cis-regulatory elements can somehow influence the Orb2 protein amount in synapses.
A model that could explain our results is as follows: The UTR sequences remaining in orb2 R transcripts lack signals critical for neuronal transport, and the levels of the orb2 mRNA in the synaptic fractions are consequently reduced. The same would be true for the orb2 tub replacement. The levels of the Orb2 protein are reduced in both orb2 R and orb2 tub , and this reduction is responsible for elevated translation of the csp and pyd mRNAs in the synaptic fraction. One would suppose that the failure to properly regulate translation in the synaptic fraction is responsible for deficits in LTM formation as well. In the case of Csp and Pyd, the protein levels increase rather than decrease. Thus, Orb2 appears to repress, rather than activate, the translation of the mRNAs encoding these two proteins. Since both mRNAs have CPEs and bind with the Orb2 protein in tissue culture cells [43], it is quite possible that Orb2 regulates their translation directly. While CPEBs are known to activate translation by promoting polyadenylation, there are contexts and mRNAs whose translation is negatively regulated by CPEBs. It will clearly be of interest to determine whether the translation of other potential Orb2 targets in neurons is repressed rather than activated.
While the distribution of the csp and pyd mRNAs in orb2 R and orb2 tub cell bodies and synaptic fractions is similar to the WT, this is not true for the actin5C mRNA. The mRNA level of the actin5C mRNA is significantly reduced in the synaptic fraction as are the levels of the orb2 mRNAs in orb2 R and orb2 tub flies. This finding raises the possibility that Orb2 may be involved in the transport of a subset of neuronal mRNAs. It will be of interest to examine the distribution of other neuronal mRNAs in orb2 R and orb2 tub .
Thus, our results allow us to assume a new function for the 3 UTR of the orb2 mRNA. In Figure 7, we summarize our hypothesis. The 3 UTR of the orb2 mRNA mediates the interaction between Orb2 proteins in the soma of a neuron. Multiple copies of connected Orb2 proteins form an RNP complex, which can more efficiently bind to transport proteins and be transported to synapses. Once in the synapse, the Orb2 protein regulates the local translation of its target mRNAs. We suppose that further studies will provide new insights into the roles of long 3 UTRs in neuronal cell functions and their participation in intracellular transport.

Figure 7.
A model of the orb2 role in neurons. The Orb2 protein participates in two major processes, intracellular transport in neurons and local translation in synapses. In the soma, Orb2 binds to the 3′UTRs of its target transcripts and participates in the formation of transport-competent mRNP. This mechanism is valid for several transcripts-e.g., Orb2 is required for intracellular transport of its own mRNA and the act5C mRNA to synapses, but not for transport of the csp and pyd transcripts. The orb2 R and orb2 tub mutants thus show lower synaptic levels of the orb2 mRNA and Orb2 protein.
In synapses, Orb2 participates in regulating translation. The orb2 R and orb2 tub mutants consequently show higher levels of csp and pyd mRNA translation in synapses. The orb2 orb mutant shows complete rescue of the described molecular defects.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure    The Orb2 protein participates in two major processes, intracellular transport in neurons and local translation in synapses. In the soma, Orb2 binds to the 3 UTRs of its target transcripts and participates in the formation of transport-competent mRNP. This mechanism is valid for several transcripts-e.g., Orb2 is required for intracellular transport of its own mRNA and the act5C mRNA to synapses, but not for transport of the csp and pyd transcripts. The orb2 R and orb2 tub mutants thus show lower synaptic levels of the orb2 mRNA and Orb2 protein. In synapses, Orb2 participates in regulating translation. The orb2 R and orb2 tub mutants consequently show higher levels of csp and pyd mRNA translation in synapses. The orb2 orb mutant shows complete rescue of the described molecular defects.