Fig 1: HRI kinase is responsible for the proteasome-inhibition induced increases in eIF2a phosphorylation.(A) Genetic deletion (KO) or inhibition of the eIF2a kinases GCN2, PERK or PKR did not rescue the inhibition of protein synthesis elicited by proteasome inhibition. (wt vs. kinase KO or inhibition: GCN2, unpaired t-test p=0.0001, PERK, unpaired t-test p=0.001 and PKR p=0.01, respectively, n = 2 experiments, two biological replicates per KO). Error bars = SD. (B) Representative puromycylation western blot showing that genetic deletion of HRI kinase rescues the inhibition of protein synthesis. Molecular weight markers from top-to-bottom are 250, 150, 100, 75, 50, 37, 25, 20, and 15 kD. (C) Analysis of the experiment shown in B. The amount of PI-induced protein synthesis inhibition was significantly reduced in the HRI KO (unpaired t-test on wt PI vs KO. PI p=0.0001, wt control vs. PI p=0.0001, KO. control vs. PI p=0.05, n = 5 experiments, error bars SD. (D) Metabolic labeling (magenta; using puromycylation, see Materials and methods) of the global nascent protein pool in cultured hippocampal neurons following proteasome inhibition showing that HRI KO neurons exhibit less PI-induced inhibition of protein synthesis. Scale bar = 100 µm. (E) Analysis of experiments like that shown in D. Protein synthesis in hippocampal neurons was significantly reduced in the HRI KO following proteasome inhibition (unpaired t-test on wt control vs. k.o. control p>0.05, wt PI vs k.o. PI p=0.01, wt control vs. PI p=0.0001, KO control vs. PI p=0.05. n = 3 experiments, wt control n = 1104, wt-PI n = 955, ko control = 1457, ko-PI n = 1052 neurons). (F) Analysis of eIF2a phosphorylation in response to proteasome inhibition. PI treatment led to a significant increase in peIF2a levels. (unpaired t-test on wt control vs. PI: p=0.01, t-test on HRI KO control vs. PI: p=0.05. two experiments –see Figure 3—figure supplement 1E. (G) RNA-seq data from hippocampal slices indicate that HRI mRNA is the most abundant amongst the 4 eIF2a kinases (H) Representative fluorescence in situ hybridization image detecting HRI mRNA in neuronal somata and dendrites. Scale bar = 20 µm for somata and 10 µm for dendrites-See Figure 3—figure supplement 2A–D.Figure 3—source data 1.Source data for Figure 3B.Figure 3—source data 2.Source data for Figure 3A, C, E, F.
Fig 2: HRI activity is constitutive owing to a low heme content in neurons.(A) In vitro eIF2a phosphorylation assay by HRI; 100 and 200 pm of hemin were added to the reaction mixture (500 ng eIF2a and 3,3 ng of HRI), inhibiting the activity of the kinase (unpaired t-test control vs hemin 100pm p=0.0001, control vs hemin 200pm p=0.01). (B) In vitro eIF2a phosphorylation assay by HRI; a constant amount of eIF2a was added for each reaction combined with increasing amounts of HRI as indicated (See Materials and methods). Increasing amounts of HRI protein lead to significantly higher levels of eIF2a phosphorylation in hippocampal lysates, compared to blood lysates (the lysates were cleared of protein by heating 10 min at 60°C) (unpaired t-test 0.05 ng HRI Blood vs hippocampus p=0.05, 0.1 ng HRI Blood vs hippocampus p=0.05, 0.33 ng HRI Blood vs hippocampus p=0.05, 66 ng HRI Blood vs hippocampus p=0.05, n = 3 experiments). (C) Western blot analysis of experiments testing whether hemin (heme complexed with Fe3+) can mitigate the effects of PI. Hemin significantly reduced the PI-induced protein synthesis inhibition. (PI vs. control, p=0.0001; PI vs. PI +hemin, p=0.01 Error bars = SD, three experiments) (D) Representative images showing that HA-HRIexpressing neurons (arrow) (green; anti-HA labeling) exhibit lower levels of protein synthesis (magenta) when compared to neighboring neurons, which do not express HA-HRI. Also shown are somata and dendrites (labeled with an anti-MAP2 antibody) and nuclei (labeled with DAPI). Scale bar = 50 µm. (E) Quantification of somatic protein synthesis levels in neurons expressing or lacking HA-HRI; HA-HRI positive neurons exhibited significantly lower levels of protein synthesis, (p=0.0001, HA negative neurons n = 852 and HA positive neurons n = 253, unpaired t-test).Figure 6—source data 1.Source data for Figure 6A, B, C, E.
Fig 3: HRI exhibits low expression under basal conditions and increased expression following proteasome inhibition.(A) Analysis for ddPCR experiments showing there is no change in HRI mRNA level following proteasome inhibition, control vs. PI, (unpaired t-test, p>0.05 n=3, error bars = SD). Samples were normalized to ribosomal RNA. (B) Quantification of immunoprecipitated HRI protein levels under control conditions or after proteasome inhibition (PI). After PI (2 hr) a significant increase in HRI protein was detected in PI samples vs. control, experiments are normalized to the untreated condition (unpaired t-test p=0.0001, n = 4 experiments, error bars = SD). (C) Scheme showing the experimental procedure, cultured neurons were labeled with S35-Met for 2 hr, washed and collected after 2 and 4 hr + / - PI. (D) Analysis of immunoprecipitated and radiolabeled HRI. Under basal conditions the HRI half-life is ˜4 hr, its degradation is blocked by PI (unpaired t-test, 2 hr control vs PI p=0.05, 4 hr control vs PI p=0.05, four experiments, error bars = SD). (E) Scheme showing the doxycycline-inducible expression of the HA-tagged HRI protein. (F) Representative images of transfected neurons (arrowheads) with the bi-directional reporter resulting in robust expression of ZsGreen (green) but near absent expression of HRI (white) in the same population of cells. Also shown are somata and dendrites (labeled with an anti-MAP2 antibody). Scale bar = 50 µm. (G) Analysis of experiments in F, showing the correlation between ZsGreen fluorescence and HA (HRI) immunolabeling in individual neurons. Dotted horizontal lines indicate the area containing 90% of the HA immunofluorescence values of ZsGreen-negative (non-transfected) neurons (5–95% percentiles), the dotted vertical line indicates the threshold between ZsGreen-negative and ZsGreen positive cells. Grey dots represent ZsGreen-negative cells, magenta dots show neurons positive for ZsGreen but negative for HA, and green dots represent neurons positive for ZsGreen and above the HA 95% percentile of the ZsGreen-negative population (n = 149). See Figure 4—figure supplement 2A.Figure 4—source data 1.Source data for Figure 4A, B, D, G.
Fig 4: Expression of transfected HRI in primary neurons -/+ proteasome inhibition, after short induction with Doxycycline (2h).Left panel shows the expression of HRI tagged on the N- terminus, and the right panel shows the expression of HRI tagged on the C-terminus. HRI expression was measured by Immunofluorescence using the HA tag. tf = transfected, PI = proteasome inhibitor.
Fig 5: HRI exhibits a codon-dependent paradoxical shift to enhanced translation following proteasome inhibition.(A) Representative polysome profile showing the effect of proteasome inhibition on translation. PI led to a dramatic shift to the monosome fraction, reflecting reduced global translation. (B) Quantification of ddPCR experiments examining the abundance of actin mRNA in monosomes and polysomes, normalized to control levels. Following PI, actin mRNA exhibited the typical shift to the monosome fraction (unpaired t-test, p=0.05, n = 2 experiments (three technical replicates per experiment). (C) Quantification of ddPCR experiments examining the abundance of HRI mRNA in monosomes and polysomes, normalized to control levels. Unlike the global RNA population, HRI mRNA exhibited a significant shift to the polysome fraction following PI, (unpaired t-test, p=0.05, n = 2 experiments three technical replicates). Error bars = SD. (D) Scheme of the HRI protein showing an abundance of many rare and extremely rare codons, consistent with HRI’s extremely low level of translation under basal conditions in neurons. HRI uses more rare codons than the 86.78% of the genes expressed in the brain (see Materials and methods). (E) Representative images showing the increased expression of codon-optimized HRI-HA in comparison with HRI-wt (Scale bar = 100 µm). (F) Scheme showing the plasmids used for the transfection shown in E, the plasmid shown in the Figure 4E was split in two parts, one expressing ZsGreen and another one expressing HRI, the plasmids were transfected in 1:5 ratio (ZsGreen:HRI) (G) Analysis of the experiments in E, showing the correlation between ZsGreen fluorescence and HA immunolabeling in individual neurons from dishes co-transfected with ZsGreen and HRI-HA either as wt sequence (HRI-HA) or codon optimized (HRIopt-HA). Dotted horizontal lines indicate the area containing 90% of the HA immunofluorescence values of ZsGreen-negative (non-transfected) neurons of the HRI-HA transfections (5–95% percentiles), the dotted vertical line indicates the threshold between ZsGreen-negative and ZsGreen positive cells. Grey and black dots are represent ZsGreen-negative (non-transfected) neurons from dishes co-transfected with HRI-HA and HRI-opt-HA, respectively. ZsGreen-positive neurons from dishes co-transfected with HRI-HA are shown in green and from dishes co-transfected with HRIopt-HA are represented in magenta. Neurons positive for ZsGreen and HA are located in the upper right quadrant (n = 2484 neurons).Figure 5—source data 1.Source data for Figure 5B, C, G.
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