RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules - PubMed (original) (raw)
RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules
N L Kedersha et al. J Cell Biol. 1999.
Abstract
In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.
Figures
Figure 1
TIA-1 and TIAR coaggregate at SGs. DU 145 cells were untreated (A–C, same field) or exposed to mild heat shock (44°C for 20 min, D–F, same field). Cells were immediately fixed and processed for two-color immunofluorescence using the TIAR-specific antibody 6E3 (A and D) and the TIA-1–specific antibody ML29 (B and E), and photographed separately. TIA-1 and TIAR colocalize at SGs (arrows), which are also visible by phase-contrast microscopy (C and F). Bar, 10 μm.
Figure 2
Recombinant TIA-1, but not hnRNP A1, accumulates at SGs in arsenite-treated COS transfectants. COS cells were transfected with plasmid vectors encoding recombinant TIA-1 (A and D) or T7-tagged recombinant hnRNP A1 (B, E, C, and F) using Superfect. After 24 h, cells were cultured in the absence (A–C) or presence (D–F) of arsenite (0.5 mM, 30 min) before processing for visualization of recombinant proteins using mAbs reactive with TIA-1 (anti-3E6, the level of expression of recombinant TIA-1 greatly exceeds that of endogenous TIA-1/R, allowing the identification of transfected cells; A and D), T7 (B and E) or endogenous TIA-1/R (C and F, anti-3E6). Paired fields (B and C and E and F) are shown. Bar, 10 μm.
Figure 3
Recruitment of HSP27 to SGs. DU145 cells were untreated (A and C) or exposed to heat shock (45°C for 20 min, B and D) and stained for both TIA-1/R (A and B, anti-3E6) and HSP27 (C and D). Bar, 10 μm.
Figure 4
TIA-1/R and poly(A)+ RNA coaggregate at SGs. Untreated (A and D), arsenite-treated and immediately fixed (0.5 mM for 1 h, B and E), or arsenite-treated as in B and E and allowed to recover in arsenite-free media for 3 h (C and F) DU145s were processed for in situ immunofluorescence to detect poly(A) (A–C), and counterstained for TIA-1/R (D–F, anti-3E6). Paired fields (A and D, B and F, C and F) are shown. Bar, 10 μm.
Figure 5
Lethal stress induces the irreversible assembly of SGs. DU145 cells were cultured in the absence (F) or presence (A–E) of arsenite (2 mM, 1 h), immediately fixed (A and D) or allowed to recover for 3 h in the absence of arsenite (B, C, and E) before processing for visualization of poly(A)+ RNA (A and B), TIA-1/R (D and E), or DNA (C and F, Hoechst dye). Bar, 10 μm.
Figure 6
PABP-I is a component of SGs. Control (A and C) or arsenite-treated (0.5 mM for 40 min, B and D) DU145 cells were fixed, permeabilized, and processed for two-color immunofluorescence using antibodies to TIA-1/R (A and B, anti-3E6) or PABP-I (C and D). Both TIA-1/R and PABP-I are clearly recruited to the SGs upon stress (B and D, paired views of same field). Bar, 10 μm.
Figure 7
Effect of wild-type and mutant recombinant eIF-2α on the assembly of TIA-1/R+ SGs. (A) COS cells were transiently cotransfected with plasmids encoding β-galactosidase and either vector alone, wild-type eIF-2α, eIF-2α (S51D), or eIF-2α (S51A) as indicated at the left of the figure. After 48 h, cells were cultured in the absence (Control, left paired panels) or presence (Arsenite, right paired panels) of arsenite (1 mM, 1 h) before fixation and processing for two-color immunofluorescence microscopy using mAbs specific for β-galactosidase (green, anti–β-galactosidase) or TIA-1 (red, TIA-1–specific antibody ML-29). In each case, paired views of the same field are presented to allow the identification of transfected and untransfected cells. Arrows in the bottom right panel point out transfected cells in which arsenite-induced assembly of SGs is prevented by the expression of eIF-2α (S51A). (B) Phosphorylation of eIF-2α is required for SG formation. Transiently transfected COS cells were treated and stained as described and were scored (at least 100 transfected cells per treatment) for the presence or absense of TIAR/ 1+ SGs as detected with mAb 3E6, and expressed as a percentage of the total cells scored per treatment. The results of 3 independent experiments were averaged. (C) Effects of mutant eIF-2α on expression of β-galactosidase. COS cells were transiently transfected with the indicated plasmids and total cell lysates were analyzed by Western blot. Upper panel, β-galactosidase; lower panel, eIF-2α.
Figure 7
Effect of wild-type and mutant recombinant eIF-2α on the assembly of TIA-1/R+ SGs. (A) COS cells were transiently cotransfected with plasmids encoding β-galactosidase and either vector alone, wild-type eIF-2α, eIF-2α (S51D), or eIF-2α (S51A) as indicated at the left of the figure. After 48 h, cells were cultured in the absence (Control, left paired panels) or presence (Arsenite, right paired panels) of arsenite (1 mM, 1 h) before fixation and processing for two-color immunofluorescence microscopy using mAbs specific for β-galactosidase (green, anti–β-galactosidase) or TIA-1 (red, TIA-1–specific antibody ML-29). In each case, paired views of the same field are presented to allow the identification of transfected and untransfected cells. Arrows in the bottom right panel point out transfected cells in which arsenite-induced assembly of SGs is prevented by the expression of eIF-2α (S51A). (B) Phosphorylation of eIF-2α is required for SG formation. Transiently transfected COS cells were treated and stained as described and were scored (at least 100 transfected cells per treatment) for the presence or absense of TIAR/ 1+ SGs as detected with mAb 3E6, and expressed as a percentage of the total cells scored per treatment. The results of 3 independent experiments were averaged. (C) Effects of mutant eIF-2α on expression of β-galactosidase. COS cells were transiently transfected with the indicated plasmids and total cell lysates were analyzed by Western blot. Upper panel, β-galactosidase; lower panel, eIF-2α.
Figure 7
Effect of wild-type and mutant recombinant eIF-2α on the assembly of TIA-1/R+ SGs. (A) COS cells were transiently cotransfected with plasmids encoding β-galactosidase and either vector alone, wild-type eIF-2α, eIF-2α (S51D), or eIF-2α (S51A) as indicated at the left of the figure. After 48 h, cells were cultured in the absence (Control, left paired panels) or presence (Arsenite, right paired panels) of arsenite (1 mM, 1 h) before fixation and processing for two-color immunofluorescence microscopy using mAbs specific for β-galactosidase (green, anti–β-galactosidase) or TIA-1 (red, TIA-1–specific antibody ML-29). In each case, paired views of the same field are presented to allow the identification of transfected and untransfected cells. Arrows in the bottom right panel point out transfected cells in which arsenite-induced assembly of SGs is prevented by the expression of eIF-2α (S51A). (B) Phosphorylation of eIF-2α is required for SG formation. Transiently transfected COS cells were treated and stained as described and were scored (at least 100 transfected cells per treatment) for the presence or absense of TIAR/ 1+ SGs as detected with mAb 3E6, and expressed as a percentage of the total cells scored per treatment. The results of 3 independent experiments were averaged. (C) Effects of mutant eIF-2α on expression of β-galactosidase. COS cells were transiently transfected with the indicated plasmids and total cell lysates were analyzed by Western blot. Upper panel, β-galactosidase; lower panel, eIF-2α.
Figure 8
Effect of HA-TIA-1ΔRRM on arsenite-induced assembly of SGs. COS cells were transiently transfected with recombinant HA-TIA-1ΔRRM, allowed to express protein for 48 h, and either untreated (A and C, paired views of same fields) or treated (B and D–F, paired views of same fields) with 0.5 mM sodium arsenite for 1 h before fixation and double staining for poly(A)+ RNA by in situ hybridization (C and D), and either HA-TIA-1ΔRRM (A, B, E) or endogenous TIAR (F) by immunofluorescence microscopy using mAbs reactive with the HA tag (A, B, and E) or endogenous TIAR (6E3). Arrows point out transfected cells. Bar, 10 μm.
Similar articles
- Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.
Sun Y, Dong L, Yu S, Wang X, Zheng H, Zhang P, Meng C, Zhan Y, Tan L, Song C, Qiu X, Wang G, Liao Y, Ding C. Sun Y, et al. FASEB J. 2017 Apr;31(4):1337-1353. doi: 10.1096/fj.201600980R. Epub 2016 Dec 23. FASEB J. 2017. PMID: 28011649 - Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.
Kedersha N, Cho MR, Li W, Yacono PW, Chen S, Gilks N, Golan DE, Anderson P. Kedersha N, et al. J Cell Biol. 2000 Dec 11;151(6):1257-68. doi: 10.1083/jcb.151.6.1257. J Cell Biol. 2000. PMID: 11121440 Free PMC article. - Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules.
Kedersha N, Chen S, Gilks N, Li W, Miller IJ, Stahl J, Anderson P. Kedersha N, et al. Mol Biol Cell. 2002 Jan;13(1):195-210. doi: 10.1091/mbc.01-05-0221. Mol Biol Cell. 2002. PMID: 11809833 Free PMC article. - Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.
Kedersha N, Anderson P. Kedersha N, et al. Biochem Soc Trans. 2002 Nov;30(Pt 6):963-9. doi: 10.1042/bst0300963. Biochem Soc Trans. 2002. PMID: 12440955 Review. - Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation.
Anderson P, Kedersha N. Anderson P, et al. Cell Stress Chaperones. 2002 Apr;7(2):213-21. doi: 10.1379/1466-1268(2002)007<0213:vstroe>2.0.co;2. Cell Stress Chaperones. 2002. PMID: 12380690 Free PMC article. Review.
Cited by
- The FXR1 network acts as a signaling scaffold for actomyosin remodeling.
Chen X, Fansler MM, Janjoš U, Ule J, Mayr C. Chen X, et al. Cell. 2024 Sep 5;187(18):5048-5063.e25. doi: 10.1016/j.cell.2024.07.015. Epub 2024 Aug 5. Cell. 2024. PMID: 39106863 - Stress granule formation helps to mitigate neurodegeneration.
Glineburg MR, Yildirim E, Gomez N, Rodriguez G, Pak J, Li X, Altheim C, Waksmacki J, McInerney GM, Barmada SJ, Todd PK. Glineburg MR, et al. Nucleic Acids Res. 2024 Sep 9;52(16):9745-9759. doi: 10.1093/nar/gkae655. Nucleic Acids Res. 2024. PMID: 39106168 Free PMC article. - Stress granules in cancer: Adaptive dynamics and therapeutic implications.
Jia Y, Jia R, Dai Z, Zhou J, Ruan J, Chng W, Cai Z, Zhang X. Jia Y, et al. iScience. 2024 Jun 22;27(8):110359. doi: 10.1016/j.isci.2024.110359. eCollection 2024 Aug 16. iScience. 2024. PMID: 39100690 Free PMC article. Review. - Genome-wide expression analysis in a Fabry disease human podocyte cell line.
Snanoudj S, Derambure C, Zhang C, Hai Yen NT, Lesueur C, Coutant S, Abily-Donval L, Marret S, Yang H, Mardinoglu A, Bekri S, Tebani A. Snanoudj S, et al. Heliyon. 2024 Jul 9;10(14):e34357. doi: 10.1016/j.heliyon.2024.e34357. eCollection 2024 Jul 30. Heliyon. 2024. PMID: 39100494 Free PMC article. - Arsenite treatment induces Hsp90 aggregatesdistinct from conventional stress granules in fission yeast.
Tomimoto N, Takasaki T, Sugiura R. Tomimoto N, et al. Microb Cell. 2024 Jul 19;11:242-253. doi: 10.15698/mic2024.07.829. eCollection 2024. Microb Cell. 2024. PMID: 39040524 Free PMC article.
References
- Antic D., Keene J. Messenger RNP complexes containing human ELAV proteinsinteractions with cytoskeleton and translational apparatus. J. Cell Sci. 1998;111:183–197. - PubMed
- Berlanga J., Herrero S., De Haro C. Characterization of the hemin-sensitive eukaryotic initiation factor 2a kinase from mouse nonerythroid cells. J. Biol. Chem. 1998;273:32340–32346. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials
Miscellaneous