Abstract
In response to stress, cells attenuate global protein synthesis but permit efficient translation of mRNAs encoding heat-shock proteins (HSPs). Although decades have passed since the first description of the heat-shock response, how cells achieve translational control of HSP synthesis remains enigmatic. Here we report an unexpected role for mitochondrial ribosomal protein L18 (MRPL18) in the mammalian cytosolic stress response. MRPL18 bears a downstream CUG start codon and generates a cytosolic isoform in a stress-dependent manner. Cytosolic MRPL18 incorporates into the 80S ribosome and facilitates ribosome engagement on mRNAs selected for translation during stress. MRPL18 knockdown has minimal effects on mitochondrial function but substantially dampens cytosolic HSP expression at the level of translation. Our results uncover a hitherto-uncharacterized stress-adaptation mechanism in mammalian cells, which involves formation of a ‘hybrid’ ribosome responsible for translational regulation during the cytosolic stress response.
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Acknowledgements
We thank J.W. Yewdell and T. Fox for critical reading of the manuscript. We also thank I. Benjamin (University of Utah) for providing Hsf1+/+ and Hsf1−/− MEFs, R. Kaufman (Sanford Burnham Medical Research Institute) for eIF2α (S/S) and eIF2α (A/A) MEFs, and T. Wandless (Stanford) for plasmids encoding the FKBP destabilizing domain. This work was supported in part by US National Institutes of Health training grants to R.A.C. and C.S.C. B.L. was supported as a recipient of the Genomics Scholar's Award from the Center for Vertebrate Genomics at Cornell. This work was primarily supported by grants to S.-B.Q. from the US National Institutes of Health (DP2 OD006449 and R01AG042400), the Ellison Medical Foundation (AG-NS-0605-09) and the US Department of Defense (W81XWH-14-1-0068).
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X.Z. and S.-B.Q. conceived the original idea and designed the experiments. X.Z. performed the majority of the experiments. X.G. conducted luciferase reporter assays. R.A.C. assisted in polysome gradient analysis. C.S.C. conducted 35S metabolic labeling assays. B.L. assisted in data interpretation. S.-B.Q. wrote the manuscript, and all authors edited the manuscript.
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Integrated supplementary information
Supplementary Figure 1 MRPL18 undergoes stress-induced alternative translation.
(a) Representative immunoblot results of HeLa cells transfected with plasmids as indicated. β-actin is used as loading control.
(b) Quantitative RT-PCR of MRPL18 (left) and HSPA1A (right) in HSF1 WT and KO cells before heat shock or 3 h after heat shock (42°C, 1 h). Error bars, s.e.m. (n = 4 cell cultures).
(c) Semi-quantitative RT-PCR of transgene MRPL18 in HeLa cells transfected with GFP or MRPL18 mutants as indicated. β-actin was used as an internal loading control. The bottom panel showed the relative levels of MRPL18 transgene normalized to β-actin mRNA.
(d) Polysome profile of HeLa cells transfected with myc-MRPL18, which shows co-localization with other mitochondrial ribosomal proteins.
Supplementary Figure 2 Heat-shock stress induces a cytoplasmic isoform of MRPL18.
(a) Schematic of MRPL18-DD fusion protein in the absence or presence of shield 1 (top panel). HeLa cells transfected with plasmids encoding MRPL18-DD were immunostained with anti-myc (green channel) and anti-Hsp60 antibody (red channel). Anti-myc signal was barely visible in the absence of 1 μM shield 1.
(b) HeLa cells transfected with plasmids encoding MRPL18-DD were immunostained before heat shock in the presence of 1 μM shield 1. Multiple fields were shown with local highlights of merged images. The right panel shows the linear scanning of pixels for all three channels.
(c) Same cells as in (B) with immunostained immediately after heat shock for 2.5 h.
Supplementary Figure 3 Heat-shock stress triggers eIF2α phosphorylation.
(a) eIF2α(S/S) and eIF2α(A/A) MEF cells were subject to heat shock stress (43°C, 1 h). Cells before heat shock and 3 h after heat shock were collected followed by immunoblotting using antibodies indicated.
(b) MRPL18-Fluc constructs were transfected into eIF2α(S/S) (left panel) and eIF2α(A/A) (right panel) MEF cells with or without heat shock. Fluc activities were either normalized to WT-Fluc or before heat shock. Error bars, s.e.m. (n = 3 cell cultures).
Supplementary Figure 4 MRPL18(cyto) incorporates into the 80S ribosome in a stress-dependent manner.
(a) Polysome profiles of HeLa cells before heat shock (blue line), 2.5-h after heat shock in the absence (red line) or presence of 10 ug ml-1 cycloheximide (CHX) (green line). The bottom panel shows the endogenous MRPL18 in each fraction.
(b) Polysome fractions as in (a) were pooled and concentrated by ultracentrifugation followed by immunoblotting using antibodies as indicated (left panel). Relative ratio of RPs in the pooled polysome fraction after heat shock is quantitated and plotted in the right panel.
Supplementary Figure 5 MRPL18 knockdown has minimal effects on global and mitochondrial translation under normal growth conditions.
(a) HeLa cells were transfected with siRNA SMARTpool targeting MRPL18, RPL5, or control for 72 h. Cells were radiolabeled with [35S]Met/Cys for times as indicated and [35S] radioactivity of trichloroacetic acid (TCA)-insoluble material was measured. Error bars, s.e.m. (n = 3 cell cultures).
(b) MEF cells stably infected with shRNA targeting MRPL18, or control were monitored by [35S] Met/Cys labeling. Error bars, s.e.m. (n = 3 cell cultures).
(c) MEF cells (left panel) and HeLa cells (right panel) were infected with lentiviruses expressing shRNA targeting Scramble or MRPL18 followed by stable selection. Cells were treated with 100 μg ml-1 cycloheximide (CHX) to inhibit cytoplasmic protein synthesis followed by [35S] metabolic labeling for 1 h. Mitochondrial translation was inhibited by adding 100 μg ml-1 chloramphenicol (CAP).
Supplementary Figure 6 Cytosolic MRPL18 promotes Hsp70 biosynthesis after heat-shock stress.
(a) HeLa cells were transfected with siRNA SMARTpool targeting MRPL18 or control for 72 h before heat shock treatment (43°C, 1 h). Cells before heat shock and 3 h after heat shock were collected followed by immunoblotting using antibodies indicated. The bottom panel showed the relative levels of Hsp70 and Hsp40 normalized to β-actin. Error bars, s.e.m. (n = 3 cell cultures, * p < 0.05 by two-tailed Student’s test).
(b) HeLa cells were infected with lentiviruses expressing shRNA targeting Scramble or MRPL18 followed by stable selection. Cells before heat shock and 3 h after heat shock were collected followed by immunoblotting using antibodies indicated. The bottom panel showed the relative levels of Hsp70 and Hsp40 normalized to β-actin. Error bars, s.e.m. (n = 3 cell cultures, * p < 0.01 by two-tailed Student’s test).
(c) MEF cells were infected with lentiviruses expressing shRNA targeting Scramble or MRPL18 followed by stable selection. Cells before heat shock and 3 h after heat shock (42°C, 1 h) were collected followed by immunoblotting using antibodies indicated. The bottom panel showed the relative levels of Hsp70 and Hsp40 normalized to β-actin. Error bars, s.e.m. (n = 3 cell cultures, * p < 0.01 by two-tailed Student’s test).
(d) MEF cells were infected with lentiviruses expressing shRNA targeting Scramble or MRPL18 followed by stable selection. Cells before heat shock and 3-h after heat shock (42°C, 1 h) were collected followed by quantitative RT-PCR for MRPL18 (left panel) and Hsp70 (right panel).. Error bars, s.e.m. (n = 4 cell cultures).
Supplementary Figure 7 Characterizing MRPL18 in the cytosolic stress response.
(a-b) HeLa cells were transfected with siRNA targeting MRPS18B, MRPL38, or control. Knockdown efficiency (a) and heat shock-induced Hsp70 synthesis (b) were examined by immunoblotting.
(c) HeLa cells were transfected with siRNA targeting RPL5 or control. Whole cell lysates were collected before and after heat shock followed by immunoblotting. * non-specific band.
(d) In vitro synthesized 5S rRNA was biotinylated followed by incubation with recombinant GFP or MRPL18(ATG) proteins with varied amounts (0, 0.1, 0.3, and 0.9 μg). After washing, the proteins bound to 5S rRNA was eluted and resolved in SDS-PAGE followed by immunoblotting using antibodies indicated.
(e) In vitro synthesized 5S rRNA was incubated with increasing amount of BSA or MRPL18(ATG) (0.1, 0.3, and 0.9 μg) followed by electrophoretic mobility shifting assay. 5.8S rRNA was included as a negative control.
(f) Ribosome subunits (40S and 60S) from HeLa cells before (left panel) and after (right panel) heat shock were separated by EDTA treatment followed by sucrose gradient separation. Immunoblotting of ribosome fractions was conducted using antibodies as indicated.
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Zhang, X., Gao, X., Coots, R. et al. Translational control of the cytosolic stress response by mitochondrial ribosomal protein L18. Nat Struct Mol Biol 22, 404–410 (2015). https://doi.org/10.1038/nsmb.3010
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DOI: https://doi.org/10.1038/nsmb.3010
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