For experiments in living cells (in primary culture of hippocampal neurons), constructs based on the p156 vector were obtained. The non-integrated lentiviral vector p156RRL (rabbit reticulocyte lysate)–sin18-PPT-PRE (Terskikh et al., 2005) was kindly provided by Alon Chen (München, Germany). These constructs contained the PKMζ 5′UTR (wild-type or mutated) fused to mCherry followed by CRPV IRES and GFP as an internal control.

The constructs for mRNAs for in vitro experiments were prepared by insertion of PKMζ or actin 5′UTRs followed by the firefly luciferase coding sequence into pGEM-T vectors. PCR based site-directed mutagenesis was used to mutate AUG codons in the PKMζ 5′UTR.

PCR templates for synthesis of mRNAs for in vitro experiments were obtained directly from the pGEM-based constructs with the corresponding T7 promoter-containing forward primers and sequence specific reverse primers (Dmitriev et al., 2007). The 50 μl transcription mixture contained 40 mM Hepes-KOH, 1 mM spermidine, 25 mM MgCl2, 1 mM DTT, 5 mM 4NTPs, 1 u/mkl Ribolock, 5 u/μl T7 RNA polymerase, and 200 ng/μl of the PCR template. The resulting transcripts were precipitated with 2 M LiCl. For all transcripts, the Vaccinia Capping System (NEB) was used to obtain 100% capped products.

Rnor 6.0 genome version of Rattus norvegicus assembly and GRCm38 genome version of Mus musculus were used. For rat genes, the 5′ UTR regions were defined as the longest regions from annotated mRNA start to the appropriate annotated CDS start. For mouse genes, annotated 5′ UTR regions were extracted. ORF in UTR regions were found by using EMBOSS getorf tool. The number of UORF for each gene or specified group of genes was counted using a homemade script. The results of computational analysis are summarized in Supplementary Figure S1.

In vitro translation was performed in Krebs-2 cells S30 extract or in RRL (Promega), in total volumes of 10 and 20 μl, respectively (Terenin et al., 2016). Reaction mixtures contained 5 μl of the S30 extract or 10 μl of the RRL, translation buffer (20 mM Hepes-KOH pH 7.6, 1 mM DTT, 0.5 mM spermidine-HCl, various amounts of Mg(OAc)₂ (1–3.5 mM), 8 mM creatine phosphate, 1 mM ATP, 0.2 mM GTP, various amounts of KOAc (40–120 mM) and 25 μM of each amino acid), 2 u of RiboLock RNase inhibitor (Thermo Scientific), 0,1 mM luciferin, 300 ng of the PKMζ 5′UTR mRNA or 75 ng of the actin 5′UTR Mrna, and in some cases 5 pmol of initiation factors (eIF1, eIF1A, eIF2, eIF4A, eIF4B, eIF4G, or ΔeIF4G). The kinetics of luciferase synthesis was measured using a Tecan Infinite200pro at 30°C for 30 min.

To assemble ribosomal complexes, we utilized commercially available RRL (Promega). We followed the previously published protocol (Dmitriev et al., 2003) with some modifications. The reaction was initiated in a total volume of 9 μl containing 7 μl RRL, 1 mM Mg2+, 0.5 u/μl Ribolock and 75–200 ng of mRNA, and was incubated for 5 min at 30°C. After that, 10 μl of RT Mix were added directly into the tube at 30°C. The RT Mix contained 20 mM Tris-HCl pH 7.5, 60 mM KCl, 0.25 mM spermidine-HCl, 1 mM DTT, 2 μl of dNTP mix (5 mM each), 1 μl of 6-carboxyfluorescein-labeled oligonucleotide (5 pmol/μl, 5′-FAM-GATGTTCACCTCGATATG-3′), 1 μl of 0.5 M Mg(OAc)₂, 1 μl of AMV Reverse Transcriptase (Promega), and 4 μl of water. The mixture was incubated for 15 min at 30°C. The resulting cDNAs were then purified by thorough phenol/chloroform extraction, precipitated with ethanol, dissolved in 100%-formamide and analyzed by capillary gel electrophoresis in an ABI PRISM 3100-Avant Genetic Analyzer (Applera).

For each test, three experimental and three control cultures were used. All experimental procedures were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) on the protection of animals used for scientific purposes. The study protocol was approved by the Ethics Committee of the Institute of Higher Nervous Activity and Neurophysiology of RAS. Wistar rat pups (P0–P2) were euthanized by decapitation with sharp scissors. The brains were removed, hippocampi dissected and gently cut into pieces with a sharp blade in ice-cold DMEM solution (Paneco) with glutamine. Next, the tissue was treated in DMEM with trypsin (10 mg in 12,5 ml of solution) for 15 min at 36°C and then centrifugated at 2000 rpm for 2 min. Cells were washed with the Neurobasal medium followed by centrifugation at 2000 rpm for 2 min two times. Then the tissue was resuspended in Neurobasal Medium (Gibco) with 2% B-27 supplement (Gibco), GlutaMax (Gibco), and cells were placed onto 12 mm glass coverslips coated with poly-D-lysine (SIGMA). Cultures were housed in a CO₂ incubator at 5% CO₂ concentration and 37°C temperature prior to the fluorescent imaging experiments.

At the seven DIV, neurons were transfected by plasmids using Lipofectamine 2000 (Invitrogen). The following day, picrotoxin (to a final concentration of 30 μM) or a control solution was added to culture. Fluorescent images were obtained at nine DIV using microscope Keyence BZ-9000 (Japan) and analyzed with ImageJ (NIH). For quantitative comparison of mCherry expression in different neurons, the fluorescence of the proximal parts of dendrites was measured. The length of measured region was 35 μm. This part was chosen for standardization of quantitative analysis. The measurement area was identical and it was located at an equal distance from the soma in all cells. The red fluorescence/green fluorescence ratio was calculated after background subtraction.

Cell cultures were prepared from neonatal rat hippocampi as described above. At the 14 DIV picrotoxin (to a final concentration of 30 μM) or control solution was added to culture. After 12 h, cells were harvested and resuspended in 1000 μl of cold (4°C) phosphate buffer saline (PBS). Next, cell suspensions were centrifuged at 1000 g, 3 min, at 4°C. The pellet was resuspended in 25 μl of cold PBS and then mixed with 25 μl of 2^x Laemmli sample buffer. Each sample was divided into two halves, in one half eIF2α and in the other p-eIF2α concentrations were detected.

Samples were incubated at 95°C for 10 min, loaded to 12% TGX Stain-Free FastCast acrylamide gel (Bio-Rad) and electrophoresed according to the supplier recommendations. Total protein levels were estimated in the gels using the ChemiDoc Touch Imaging System (Bio-Rad), Stain-Free assay protocol. Proteins were then transferred from gels to PVDF membranes by electrophoresis with the standard program for TGX gel for 3 min. The SNAP i.d.^® 2.0 Protein Detection System (Millipore) was used to shorten the time required for blocking, washing, and antibody incubations. After blocking with 0.5 % no fat milk (in PBS+0.1% tween-20, 30 ml) for 10 min, membranes were incubated in 5 ml of primary antibodies solution [rabbit antibodies to eIF2α (Cell Signaling, #5324) or p-eIF2α (Cell Signaling, #3398) diluted 1:1000 in milk] for 10 min, then washed with 120 ml of PBS+tween for 5 min. For incubation with secondary antibodies [goat anti-rabbit conjugated with horseradish peroxidase (Bio-Rad, #172–1019), diluted 1:1000 in milk] and their washing the same protocol as for primary antibodies was used.

Bands were detected with the ECL Advance Western blotting detection kit (GE Healthcare) on the ChemiDoc Touch Imaging System, chemiluminescence assay. Immunoblot signals were quantified by densitometric scanning and image analysis with Image Lab Software (Bio-Rad). Optical density of eIF2α and p-eIF2α bands was normalized to total protein in the lane. After normalization, the p-eIF2α/eIF2α ratio was calculated for each sample. Samples obtained from four independent experiments.

To examine the effect of the structure and composition of 5′UTR of PKMζ mRNA on its translation, we designed genetic constructions containing substitutions of start codon ATG to stop codon TAG of each uORF in 5′UTR. Thus, we “turned off” all seven uORFs of 5′UTR PKMζ in consecutive order (Figure 1A). As a result, we obtained seven genetic constructions that had the same length but contained different numbers of uORFs. The modeled secondary structures of “wild-type” 5′UTR and Δ1–7 construct, with disabled uORFs, have the same forms with similar energies -227 ccal/mole (Figure 1B). This means that “turning off” the uORFs does not significantly change the overall structure of mRNA. To estimate the efficiency of translation of these constructions in vitro (in cell lysates), the mutated 5′UTRs were inserted into pGEM-T plasmid encoding luciferase. The efficiency of translation of the obtained mRNAs was detected in RRL (Figure 1C) and mouse ascite carcinoma cell lysate (S30; Figure 1D).

We found that translation of the main ORF was more effective if 5′UTR contained a reduced numbers of uORFs. This result corresponds to the concept of translation inhibition by uORFs. It was shown previously (Hernandez et al., 2003) that shortening the 5′UTR of PKMζ increased luciferase translation in vitro. This may be caused by reduction in the number of uORFs or by changes in mRNA secondary structure (unfolding; Hernandez et al., 2003; Barbosa et al., 2013). Our data demonstrate that the translation rate depends on the presence of uORFs that compete for translation initiation complexes with the main coding frame, and is not affected by the secondary structure of the leader sequence.

We demonstrated the importance of uORFs for the translational control of PKMζ. The question then arises whether this mechanism of regulation is a basic one for neurons. To answer the question we performed bioinformatics analysis of the available soma and dendritic associated transcriptome data sets for mouse (Ainsley et al., 2014). In this research, only neuronal mRNAs from hippocampal CA1 region were investigated. We conducted a comparison of neuronal and non-neuronal whole transcriptome data sets (Supplementary Figure S1A). The neuronal data set combines soma and dendritic associated transcriptomes. In this case, we observed a difference in the percentage of expressed genes, which was dependent on the number of uORFs in mRNA. The percentage of expressed genes with 0–2 uORFs is smaller in the neuronal transcriptome than in the whole transcriptome. On the contrary, the amount of expressed genes with 3–10 uORFs is greater in the neuronal fraction compared to the whole transcriptome (p-value < 0.0001, Chi square test). This result suggests that regulation of translation by uORFs is widely used in neurons.

Moreover, we carried out a comparison of the neuronal transcriptome and Ribo-seq data obtained 5 min after learning of M. musculus whole hippocampus (the sample contains neuronal and non-neuronal cells; Cho et al., 2015) (Supplementary Figure S1B). We determined that after learning the amount of translated mRNAs containing a large number of uORFs increases, and the amount of translated mRNAs without or with small number of uORFs decreases (p-value < 0.0287, Chi square test). This indicates a possible involvement of uORFs into translational control during memory consolidation.

Another possibility for regulation of the translation efficacy may be a control of the level of ion concentrations in cells. As translation of PKMζ in non-activated neurons is suppressed, we decided to determine the contributions of the physiological concentrations of K⁺ and Mg²⁺ into such inhibition. We measured the efficiency of translation of model mRNA, which contains 5′UTR of PKMζ and the luciferase coding sequence, as a function of the concentration of cations (both K⁺ and Mg²⁺) in the cell lysate (Figures 2A,B). As a control, mRNA with 5′UTR of actin followed by luciferase CDS was used. We observed that the optimal cation concentrations for translation of mRNA with PKMζ 5′UTR were very narrow.

The efficiencies of translation of both mRNAs in 1 mM Mg²⁺ were identical and maximal (Figure 2A). However, increasing of the magnesium concentration to 3 mM lead to significant suppression of translation of mRNA with PKMζ 5′UTR. The translation of the control (actin) mRNA remained high. Interestingly, concentrations of Mg²⁺ in dendrites may vary (Cheng and Reynolds, 2000; Palacios-Prado et al., 2014). There may be local oscillations of Mg²⁺ concentration in neurons between 0.2 and 3.5 mM due to the reversible ATP-Mg²⁺ binding (Palacios-Prado et al., 2014). Therefore, different energy-consuming intracellular processes, including action potential generation or postsynapse activation, may cause an increase of the local Mg²⁺ concentration because of ATP consumption.

A similar effect was observed with various concentrations of K⁺ in the translation mixture (Figure 2B). If the K⁺ concentration in the solution was equal to the normal intracellular level (140 mM; Müller and Somjen, 2000), the translation efficacy was low. It constituted only 15% of the efficacy level that was observed if there was 80 mM K⁺ in the solution (Figure 2B). Translation of the control mRNA was slightly influenced by the potassium concentration. Consequently, the translation of PKMζ in normal concentrations of K⁺ and Mg²⁺ (140 and 2,5 mM, respectively) is suppressed and requires additional activation.

The 5′UTR of PKMζ is 600 nt long and very structured. We have already shown that disabling uORFs does not affect its structure (Figure 1B). To confirm the high complexity of this sequence we applied the toe-printing analysis of mRNA with firefly luciferase coding sequence and 5′UTR of PKMζ in RRL (Figure 3A). The translation initiation complexes assembled on the mRNA in cell lysate could be detected by reverse transcription with the fluorescently labeled primer complimentary to the 3′-end of the RNA. The lengths of obtained cDNA fragments reflect the positions of ribosomal complexes or hairpins on the mRNA. We observed that the leader of PKMζ has many hairpins near the main ORF that prevent the movement of reverse transcriptase in the toe-printing assay. Furthermore, we didn’t detect any translation initiation complex on the main ORF using mRNA with 5′UTR of PKMζ. As a control we used mRNA with the same firefly luciferase coding sequence and short (86 b.) uORF-free 5′UTR of actin (Figure 3A). This mRNA had no hairpins and efficiently assembled the translation initiation complex on the ATG codon of the main frame. Therefore, we showed that the formation of the initiation complex on the start codon of the main frame in the presence of 5′UTR of PKMζ is strongly suppressed.

It is known that the eukaryotic initiation factor eIF4B amplifies helicase-like activity of eIF4A and it is necessary for translation of mRNAs with a complex secondary structure of 5′UTRs (Andreou and Klostermeier, 2013). To obtain additional data about the effect of the structure of 5′UTR on translation of PKMζ, we incubated eukaryotic initiation factors eIF4A and eIF4B in the RRL cell-free translation system with the wt and Δ1–7 mRNAs (Figures 2C,D). These factors demonstrated a moderate stimulatory effect on the efficiency of translation on both mRNAs. It also confirms the hypothesis that the structure of 5′UTR of PKMζ does not contribute significantly to its translation (see above).”

The truncated eIF4G (ΔeIF4G, p50) consists of the central third of eIF4G (aa 486–935) which interacts with both eIF3 and eIF4A and lacks PABP and eIF4E binding sites (De Gregorio et al., 1998). An addition of ΔeIF4G stimulates translation of 5′UTR of PKMζ (Figure 2E). On the contrary, this protein suppresses the translation of Δ1–7 mRNA. This is consistent with the earlier studies where p50 exerted (Figure 2F) a dominant negative effect on the translation of capped mRNAs and stimulated the translation of uncapped mRNAs (De Gregorio et al., 1998). Moreover, p50 strongly enhanced re-initiation in the eIF4G-depleted lysates (Pöyry et al., 2004).

Our results suggest that uORFs play the main role in the suppression of translation of PKMζ mRNA. To confirm our suggestion, we performed a toe-printing analysis of two mutant 5′UTRs of PKMζ, with AUG substituted by UAG in the first or second uORF (Figure 3B). On the wt 5′UTR of PKMζ, the 48S initiation complex assembles mainly on the first uORF, and we didn’t observe any ribosomal complex on AUG codon of the main ORF (Figure 3A). If the AUG codon of the first uORF was mutated (Δ1), 48S complexes were not registered in this position, but some complexes were shown to be located on the second uORF. On the other side, if the AUG codon of the second uORF was mutated (Δ2), initiation complexes were detected only on the first uORF (Figure 3B). Thus, we showed that uORFs within 5′UTR of PKMζ are indeed able to assemble the translation initiation complexes, and these uORFs may affect the translation from the main coding frame.

To investigate the PKMζ translation control in living cells we used a bicistronic system of two reporter fluorescent proteins that were inserted to the same plasmid (Figure 4A). The translation of the first protein, mCherry, was controlled by 5′UTR (wild-type or mutant) and 3′UTR of PKMζ. The second encoded protein, GFP, was controlled by the CPV-IRES-element that allows the 40S and 60S subunits to form 80S initiation complex without translation initiation factors and start translation immediately (Figures 4B,C) (Pestova et al., 2004). Therefore, the GFP translation was constitutive, and GFP fluorescence can be used for signal normalization.

On Figure 5A the normalized mCherry/GFP fluorescence level ratios in cell cultures transfected by plasmids with or without uORFs in PKMζ 5′UTR are shown. We found that the patterns of fluorescence were different in the cells transfected by plasmids containing wild-type or mutant 5′UTR of PKMζ (Figures 4B,C). In the case of the wild-type 5′UTR, synthesis of mCherry in dendrites was suppressed. On the contrary, the basic level of mCherry translation was high in the culture transfected by mutant Δ1-7 5′UTR of PKMζ. Using quantitative comparison (Figures 5A,B), we determined a twofold difference between fluorescence in the proximal parts of dendrites containing wt and mutant 5′UTRs of PKMζ (p < 0,00001, N = 80–86 neurons from three independent experiments). The changes in translation of mCherry caused by neuronal activation (we used picrotoxin to activate the cells) were also different in these two cultures. Namely, after activation the mCherry signal increased in wild-type PKMζ 5′UTR-containing neurons (p < 0,001, N = 58 neurons from three PTX-treated cultures), but didn’t change significantly in the cells that were transfected by plasmid carrying Δ1–7 5′UTR of PKMζ (p > 0,05; N = 42 from three independent cultures). Since mCherry fluorescence level correlates to the mCherry protein accumulation, we proved that uORFs are crucial for PKMζ translation. We also confirmed that translation of mRNA with wild-type 5′UTR of PKMζ increases after synaptic activation.

We suppose that the demonstrated increase of translation of mRNA with PKMζ regulatory elements in response to synaptic activation was caused by the eIF2α phosphorylation, which also occurs during cellular stress (Harding et al., 2000). To verify this hypothesis, we induced mild cellular stress by sodium arsenite in the cell cultures transfected by plasmids with or without uORFs in PKMζ 5′UTR (Figure 5C). Earlier it was shown that sodium arsenite induced stress is characterized by eIF2α phosphorylation and global translation arrest (McEwen et al., 2005; Tsai et al., 2009; Andreev et al., 2015). Our experiments showed that in the proximal parts dendrites of neurons containing the wild-type 5′UTR of PKMζ, the arsenite-induced stress causes a tendency for the relative fluorescence to increase (p < 0,1; N = 36–40 neurons in control and arsenite-treated group). In neurons containing the mutant 5′UTR of PKMζ, arsenite didn’t cause any significant changes in relative fluorescence (p > 0,05; N = 30–63 neurons). Therefore, we have concluded that translation of PKMζ mRNA in neurons may be associated with the eIF2α phosphorylation.

To further confirm our suggestion, we needed to check the level of eIF2α phosphorylation after activation of cells with picrotoxin. For this purpose, we determined the levels of the phosphorylated eIF2α (p-eIF2α) and non-phosphorylated eIF2α by immunoblotting in the same primary cell culture before and after picrotoxin activation. We found that the p-eIF2α/eIF2α concentration ratio is increased in the cells exposed to picrotoxin compared to a control culture that wasn’t activated (Figures 5D,E). Therefore, we have shown phosphorylation of eIF2α after the picrotoxin-induced activation of neurons, which confirms the supposed p-eIF2-dependent mechanism of activation of PKMζ translation.