Characterization of the Role of eIF4G in Stimulating Cap- and IRES-Dependent Translation in <i>Aplysia</i> Neurons

<div><p>The rate-limiting step(s) of translation in the nervous system have not been clearly identified. We have been examining this question in the cell body of the <i>Aplysia</i> sensory neuron, where translational regulation is important for the regulation of synaptic strength. In the present study, we examined the role of the adaptor protein eIF4G. We cloned <i>Aplysia</i> eIF4G (Ap4G) and Ap4G contains all the standard metazoan eIF4G protein–protein interaction domains. Overexpressing Ap4G in <i>Aplysia</i> sensory neurons caused an increase in both cap-dependent and internal ribosome entry site (IRES)-dependent translation using a previously characterized bicistronic fluorescent reporter. Unexpectedly, measurement of overall translation using the methionine analog, L-azidohomoalanine, revealed that overexpression of Ap4G did not lead to an increase in overall translation rates. Indeed, the effect of Ap4G on the bicistronic reporter depended on the presence of an upstream open reading frame (uORF) in the 5’ UTR encoded by the vector. We have previously shown that Mnk strongly decreased cap-dependent translation and this depended on a putative 4G binding domain. Here we extend these results showing that even in the absence of the uORF, overexpression of Mnk strongly decreases cap-dependent translation and this depends on the Mnk binding site in eIF4G. Similarly, an increase in cap-dependent translation seen with overexpression of elongation factor 2 kinase did not depend on the uORF. Overall, we show that eIF4G is rate limiting for translation of an mRNA encoding an uORF, but is not generally a rate-limiting step for translation.</p> </div

Overexpressing Ap4G in <i>Aplysia</i> neurons increases cap- and IRES-dependent translation from a reporter construct.

<p>Cultured <i>Aplysia</i> sensory neurons were co-injected with a bicistronic fluorescent reporter and either empty expression vector (Con) or Ap4G (4G). The photos are representative neurons, 48 hours later; the top row shows cyan fluorescence used as a reporter of cap-dependent translation (Cap), the middle row shows yellow fluorescence used as a reporter of IRES-dependent translation (IRES) and the bottom row shows red fluorescence from immunostaining for eIF4G (4G ICC) in the same neurons. The histogram of means (normalized to the respective mean of control cells) and SEMs shows the fluorescent protein expression from the groups of the representative neurons. The ratio of IRES- to cap-dependent translation was calculated (IRES/Cap). The values, SEM and Ns are: control (Cap 1.00 ± 0.07, IRES 1.00 ± 0.05, IRES/CAP 1.00 ± 0.04, 4G 1.00 ± 0.04, n=159 neurons from 16 experiments for CAP, IRES and IRES/CAP, n= 85 neurons from 10 experiments for 4G) eIF4G (Cap 1.62 ± 0.13, IRES 1.84 ± 0.14, IRES/CAP 1.29 ± 0.16, 4G 1.90 ± 0.06, n=180 neurons from 16 experiments for CAP, IRES and IRES/CAP, n=88 cells for 4G) Student’s <i>t</i> test p values (with Welch’s and Bonferroni’s corrections) between control and eIF4G expression are shown over the bars.</p

Overexpressed Ap4G only increases translation in the context of an upstream open reading frame (uORF).

<p><b>A</b>) Cultured <i>Aplysia</i> sensory neurons were injected with either empty expression vector (Con) or Ap4G (4G). After 48 hours, overall translation was measured using AHA. In 3 of the 8 experiments, AHA labeling was done 12 hours after expression, but as these results were not significantly different than the results at 48 hrs, the results were combined into one group. Representative neurons show red fluorescence from incorporated AHA (AHA) and far red fluorescence from immunostaining for eIF4G (4G ICC) in the same neurons. Histogram is the means (normalized to Con) and SEMs from the groups of the representative neurons. The values, SEM and Ns are: Con (AHA 1.00 ± 0.04, n=98 neurons from eight experiments; 4G ICC 1.00 ± 0.02, n=102 neurons from eight experiments); eIF4G (AHA 0.67 ± 0.04, n=101 neurons from eight experiments, 4G ICC 2.87 ± 0.11, n-102 neurons from eight experiments.) P values from Student’s <i>t</i> test compared to control (with Welch’s and Bonferroni’s corrections) are shown over the bars. <b>B</b>) Deletion of uORF from bicistronic reporter construct. Beginning of eCFP ORF in italics, nucleotides that match the Kozak initiating consensus sequence are underlined and uORF is shaded. <b>C</b>) Cultured <i>Aplysia</i> sensory neurons were injected with either the original bicistronic construct (with uORF, C _IRESY) or the modified bicistronic construct (uORF deleted, ΔuORF- C _IRESY). Representative neurons (48 hours later) show cyan fluorescence (cap-dependent translation, Cap) and yellow fluorescence (IRES-dependent translation, IRES) in the same neurons. Histogram is the means (normalized to C _IRESY) and SEMs from the groups of the representative cells: The ratio of IRES- to cap-dependent translation was calculated (IRES/Cap). The values, SEM and Ns are: C _IRESY (Cap 1.00 ± 0.14, IRES 1.00 ± 0.15, IRES/CAP 1.00 ± 0.07, n=42 neurons from four experiments); ΔuORF- C _IRESY (Cap 3.47 ± 0.76, IRES 1.62 ± 0.33, IRES/CAP 0.60 ± 0.07, n=52 neurons from four experiments. P values from Student’s t-test compared to C _IRESY (with Welch’s and Bonferroni’s corrections) are shown over the bars. <b>D</b>) Cultured <i>Aplysia</i> sensory neurons were co-injected with the modified bicistronic fluorescent reporter (uORF deleted) and either empty expression vector (Con) or Ap4G (4G). Representative neurons (48 hours later) show cyan fluorescence (cap-dependent translation, Cap), yellow fluorescence (IRES-dependent translation, IRES) and red fluorescence from immunostaining for eIF4G (4G ICC) in the same neurons. Histogram is means (normalized to Con) and SEMs from the groups of the representative neurons. The ratio of IRES- to cap-dependent translation was calculated (IRES/Cap). The values, SEM and Ns are: Con (CAP 1.00 ± 0.09, IRES 1.00 ± 0.09, IRES/CAP 1.00 ± 0.04, 4G ICC 1.00 ± 0.01, n=122 neurons from twelve experiments); 4G (CAP 1.15 ± 0.12, IRES 1.28 ± 0.11, IRES/CAP 1.24 ± 0.06, 4GICC 2.07 ± 0.08, n=114 neurons from twelve experiments) P values from Student’s <i>t</i> test compared to control (with Welch’s and Bonferroni’s corrections) are shown over the bars.</p

Overexpressing Ap4G stabilizes overexpressed Ap4E, but Ap4E does not contribute to increase in cap-dependent translation seen with Ap4G.

<p><b>A</b>) Cultured <i>Aplysia</i> sensory neurons were injected with either empty expression vector (Con), Ap4G (4G), Ap4E (4E), Ap4E plus Ap4G (4E+4G) or Ap4E plus Ap4G with mutated eIF4E-binding site (4E+4GΔ4E). Representative neurons show red fluorescence from immunostaining for eIF4E, 48 hours later. Histogram is means (4G normalized to Con; 4E+4G and 4E+4GΔ4E normalized to 4E) and SEMs of the groups of the representative neurons. The values, SEM and Ns are: endogenous 4E, (control 1.00 ± 0.03, n=49 neurons from three experiments; 4G 0.87 ± 0.03, n=51 cells from three experiments) overexpressed 4E (4E 1.00 ±0.08, n=33 neurons from three experiments, 4E+4G 1.71 ± 0.12, n=36 neurons from three experiments, 4E + 4Gd4E 1.20 ± 0.16, n=42 neurons from three experiments. The experiments with endogenous 4E were from a subset of the experiments displayed in Figure 2. Student’s <i>t</i> test p values (with Welch’s and Bonferroni’s corrections) are shown over the bars for the comparison between control and 4G. For the comparison between control (4E), 4E + 4G, and 4E + 4Gd4E a non-parametric Kruskal-Wallis ANOVA was performed (KW statistic =22.6, p<0.001]. Dunn’s post Hoc tests showed that 4E + 4G is different from all other groups (both p<0.01, * in figure) but 4E is not significantly different from 4E + 4Gd4E (p >0.05). <b>B</b>) Cultured <i>Aplysia</i> sensory neurons were co-injected with a bicistronic fluorescent reporter and either empty expression vector (Con), Ap4E (4E), Ap4G (4G) or Ap4E plus Ap4G (4E+4G). Representative neurons (48 hours later) show cyan fluorescence (cap-dependent translation, Cap) and yellow fluorescence (IRES-dependent translation, IRES) in the same neurons. Histogram is means (normalized to Con) and SEMs from the groups of the representative cells. The ratio of IRES- to cap-dependent translation was calculated (IRES/Cap). The values, SEM and Ns are: Con (Cap 1.00 ± 0.10, IRES 1.00 ± 0.08, IRES/CAP 1.00 ± 0.07, n=54 cells from 4 experiments_; 4E (Cap 0.77 ± 0.09, IRES 0.90 ± 0.09, IRES/CAP 1.15 ± 0.11, n=56 cells from four experiments); 4G (Cap 1.53 ± 0.17, IRES 1.89 ± 0.23, IRES/CAP 1.16 ± 0.09, n=66 cells from four experiments); 4E + 4G (Cap 1.33 ± 0.11; IRES 1.24 ± 0.12; IRES/CAP 0.82 ± 0.07, n=53 cells from four experiments. These experiments represent a subset of the experiments used to calculate the effect of eIF4G vs Control in Figure 2. For the comparison between groups a non-parametric Kruskal-Wallis ANOVA was performed for CAP, IRES and the IRES/CAP ratio and then Dunn’s post-Hoc tests were performed to see if groups were different than control: Cap (KW statistic = 26.4, p<0.001. Both 4G and 4E + 4G were different than control, *, p<0.05); IRES (KW statistic = 16.6, p<0.001, Only 4G was significantly different than control *, p<0.05); CAP/IRES (KW statistic =8.84, p>0.05).</p

Increase in membrane excitability with 5HT in three different sized animals.

<p>(A) Counts of the number of action potentials during 500ms depolarizing pulses of varying current amplitudes in a representative pleural sensory neuron from an animal of approximately 100g. The slope of this relationship is a more sensitive indicator of changes in membrane excitability than measuring action potential threshold alone. The increase in excitability of the sensory neuron with 5HT can be measured from the change in the slope. (B) Sensory neurons isolated from an animal of three distinct sizes, small 78g, a larger animal 225g and a very small animal at 23g. A significant increase in excitability measured with the number of action potentials per nA of depolarizing current was observed at sensory neurons from all three animals (Comparing before and 5HT with a two-way ANOVA with Bonferroni post tests at all three animal weights, *P<0.05, **P<0.01). Data from sensory neurons from single animals in each group (n = 6 sensory neurons examined from each animal). A one-way ANOVA of the initial slopes (before) found no significant difference between the three animals.</p

eEF2 kinase overexpression selectively inhibits IRES dependent translation independently of the uORF.

<p>Cultured <i>Aplysia</i> sensory neurons were injected with either empty expression vector (Con) or <i>Aplysia</i> eEF2K (eEF2K). Representative neurons (48 hours later) show cyan fluorescence (cap-dependent translation, Cap) and yellow fluorescence (IRES-dependent translation, IRES) in the same neurons. Histogram is the means (normalized to Con) and SEMs from the groups of the representative neurons. The ratio of IRES- to cap-dependent translation was calculated (IRES/Cap). Values, SEMS and ns are: Control (CAP 1.00 ± 0.09, IRES 1.00 ±0.07, IRES/CAP 1.00 ± 0.06, n =51 neurons from four experiments); eEF2K (CAP 1.28 ± 0.17, IRES 0.35 ± 0.02, IRES/CAP 0.35 ± 0.04, n=52 neurons from four experiments. P values from Student’s <i>t</i> test compared to control (with Welch’s and Bonferroni’s corrections) are shown over the bars.</p

eGFP-PKC Apl II translocation and recovery from synaptic depression with PDBu from animals >120g.

<p>(A) Representative confocal images of a sensory neuron soma from an animal >120g expressing eGFP-PKC Apl II before and after application of 200nM PDBu. While 10μM 5HT did not result in an increase in the F_mem/F_cyto ratio, 200nM PDBu significantly increased the F_mem/F_cyto ratio at sensory neurons from >120g animals (comparing F_mem/F_cyto before to after with a two-way ANOVA and Bonferroni post tests, 5HT P>0.05, PDBu P<0.05, n = 6 for each group). (B) In a second experiment, also with sensory neurons isolated from animals >120g, 5HT then PDBu were added sequentially with an image before, after 5min in 5HT, and after another 2min in 5HT + PDBu. Again, in this group of sensory neurons, 5HT had little effect on the eGFP-PKC Apl II translocation, however, the subsequent application of PDBu significantly increased translocation (P<0.05 comparing F_mem/F_cyto with 5HT to PDBu with a paired t-test, data from 36 sensory neurons in n = 10 dishes). C) Homosynaptic depression of PSP amplitude and recovery from depression with 5HT or PDBu at synapses formed with sensory neurons from animals >120g. PDBu significantly increases facilitation following depression over 5HT (comparing the data sets with a two-way ANOVA and Bonferroni posttests, PSP1-PSP40 P>0.05 and PSP41-PSP52 P<0.001, n = 9 for 5HT and n = 7 for PDBu). Scale bars are 20<b>μ</b>m.</p

Correlation between the weight of the animal and synaptic depression or subsequent facilitation with 5HT.

<p>(A) The amount of facilitation following homosynaptic depression (PSP41 as a percentage of PSP1) was significantly correlated with the weight of the animal the sensory neurons were isolated from (Pearson r = -0.4087, 107 synapses with results averaged for each animal, n = 47 animals, P<0.01). (B) The amount of depression (the amplitude of PSP40 as a percentage of PSP1) did not correlate with the weight of the animal (Pearson r = -0.1973, n = 47, P = 0.19). Results from the individual recordings plotted as black triangles (but averaged for each animal for statistical analysis) and grey boxes are 25g binned averages in both A&B. (C) For thirty of the synapses recorded in A and B, the age of the animals from hatching dates are known. When the amount of facilitation with 5HT following synaptic depression is compared with the post-hatching age of the animal the sensory neurons were isolated from there is no correlation (Pearson r -0.2943, n = 12 animals, P = 0.33). (D) Bar graph of the amount of recovery from synaptic depression with 5HT grouped according to the month the experiment was conducted. There is no significant difference in the recovery from depression and the month of the experiment as measured with a one-way ANOVA, P = 0.1, same data set presented in A&B.</p

Translocation of eGFP-tagged PKC Apl II with 5HT in sensory neurons from animals <80g or >120g.

<p>(A) Representative confocal images of eGFP-PKC Apl II before and after 5min in 10μM 5HT in sensory neuron somata in culture from an animal <80g. The ratio of the eGFP fluorescence intensity at the membrane (F_mem) to the cytosol (F_cyto) is greatly increased with 5HT. F_mem/F_cyto was significantly increased with 5HT, P<0.05, (15 sensory neurons from n = 7 different animals). (B) eGFP-PKC Apl II translocation from the cytosol to the plasma membrane was not observed with sensory neurons from animals larger than 120g. The F_mem/F_cyto was not significantly changed with 5HT measured at 5min compared to before 5HT, P>0.05. (11 sensory neurons from n = 5 different animals). Data in AB compared with a two-way ANOVA and Bonferroni post tests. (C) Comparison of the change in eGFP-PKC Apl II translocation with 5HT in sensory neurons from <80g or >120g animals. The F_mem/F_cyto ratio with 5HT is normalized as a percent before 5HT. Scale bars are 20<b>μ</b>m.</p

Reduction in 5HT-mediated recovery from synaptic depression with sensory neurons from animals larger than 120g.

<p>(A) Voltage traces of sensory neuron action potential evoked PSPs recorded in the motor neuron at synaptic connections with the sensory neuron isolated from an animal less than 80g (top traces) and greater than 120g (bottom traces). Only traces of PSP1 (first PSP), PSP40 (40th PSP, depressed PSP), and PSP41 (41st PSP, recovery from depression with 5HT) are presented. Scale bars are 10/8mV and 10ms. (B) Time course of the change in PSP amplitude with 0.05Hz stimulation, resulting in depression, and subsequent recovery from depression with 5HT after PSP40. Use of sensory neurons from animals greater than 120g results in significantly less facilitation/recovery from depression with 5HT (Comparing the two data sets with a two-way ANOVA and Bonferroni posttests, PSP1-PSP40 P>0.05 and PSP41-PSP52 P<0.001 at all PSPs except PSP46 at P<0.01, n = 5 synaptic connections with small animal sensory neurons and n = 7 synaptic connections with sensory neurons from large animals). (C) Using sensory neurons from animals <80g, varying the size of the animal from which the postsynaptic motor neurons were isolated had no effect on the recovery from synaptic depression with 5HT. Data are from three synapses in each group. (D) The amount of depolarizing current injected into the sensory neuron to produce an action potential is reduced following the addition of 5HT (presented as the amount of current required to fire the action potential to produce PSP41 as a percentage of the current required to fire the action potential to produce PSP40). The reduction in the stimulating current in the sensory neuron was equally observed with both sensory neurons of <80g and >120g animals.</p