BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. RESULTS: We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. CONCLUSION: Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.

Centre for Genomic Regulation The Barcelona Institute of Science and Technology Dr Aiguader 88 08003 Barcelona Spain

Laboratory of RNA Biochemistry Department of Genetics and Microbiology Faculty of Science Charles University Prague Viničná 5 128 00 Prague 2 Czech Republic

Zobrazit více v PubMed

Jackson RJ, Hellen CUT, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Bio. 2010;11(2):113–127. doi: 10.1038/nrm2838. PubMed DOI PMC

Niedzwiecka A, Marcotrigiano J, Stepinski J, Jankowska-Anyszka M, Wyslouch-Cieszynska A, Dadlez M, Gingras AC, Mak P, Darzynkiewicz E, Sonenberg N, et al. Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. J Mol Biol. 2002;319(3):615–635. doi: 10.1016/S0022-2836(02)00328-5. PubMed DOI

Hou JQ, Lam F, Proud C, Wang SD. Targeting mnks for cancer therapy. Oncotarget. 2012;3(2):118–131. doi: 10.18632/oncotarget.453. PubMed DOI PMC

Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC, Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5′-cap function. Nature. 1994;371(6500):762–767. doi: 10.1038/371762a0. PubMed DOI

Dostie J, Ferraiuolo M, Pause A, Adam SA, Sonenberg N. A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5′ cap-binding protein, eIF4E. EMBO J. 2000;19(12):3142–3156. doi: 10.1093/emboj/19.12.3142. PubMed DOI PMC

Ptushkina M, von der Haar T, Karim MM, Hughes JM, McCarthy JE. Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state. EMBO J. 1999;18(14):4068–4075. doi: 10.1093/emboj/18.14.4068. PubMed DOI PMC

Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK. Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol Cell. 1999;3(6):707–716. doi: 10.1016/S1097-2765(01)80003-4. PubMed DOI

Ferrero PV, Layana C, Paulucci E, Gutierrez P, Hernandez G, Rivera-Pomar RV. Cap binding-independent recruitment of eIF4E to cytoplasmic foci. Biochim Biophys Acta. 2012;1823(7):1217–1224. doi: 10.1016/j.bbamcr.2012.03.013. PubMed DOI

Lee HC, Cho H, Kim YK. Ectopic expression of eIF4E-transporter triggers the movement of eIF4E into P-bodies, inhibiting steady-state translation but not the pioneer round of translation. Biochem Biophys Res Commun. 2008;369(4):1160–1165. doi: 10.1016/j.bbrc.2008.03.017. PubMed DOI

Yi T, Papadopoulos E, Hagner PR, Wagner G. Hypoxia-inducible factor-1alpha (HIF-1alpha) promotes cap-dependent translation of selective mRNAs through up-regulating initiation factor eIF4E1 in breast cancer cells under hypoxia conditions. J Biol Chem. 2013;288(26):18732–18742. doi: 10.1074/jbc.M113.471466. PubMed DOI PMC

Hernandez G, Altmann M, Sierra JM, Urlaub H, Diez del Corral R, Schwartz P, Rivera-Pomar R. Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila. Mech Dev. 2005;122(4):529–543. doi: 10.1016/j.mod.2004.11.011. PubMed DOI

Keiper BD, Lamphear BJ, Deshpande AM, Jankowska-Anyszka M, Aamodt EJ, Blumenthal T, Rhoads RE. Functional characterization of five eIF4E isoforms in Caenorhabditis elegans. J Biol Chem. 2000;275(14):10590–10596. doi: 10.1074/jbc.275.14.10590. PubMed DOI

Rhoads RE. eIF4E: new family members, new binding partners, new roles. J Biol Chem. 2009;284(25):16711–16715. doi: 10.1074/jbc.R900002200. PubMed DOI PMC

Hernandez G, Proud CG, Preiss T, Parsyan A. On the diversification of the translation apparatus across eukaryotes. Comp Funct Genomics. 2012;2012:256848. PubMed PMC

Friday AJ, Keiper BD. Positive mRNA Translational Control in Germ Cells by Initiation Factor Selectivity. BioMed Res Int. 2015;2015:327963. doi: 10.1155/2015/327963. PubMed DOI PMC

Nousch M, Eckmann CR. Translational control in the Caenorhabditis elegans germ line. Adv Exp Med Biol. 2013;757:205–247. doi: 10.1007/978-1-4614-4015-4_8. PubMed DOI

Joshi B, Lee K, Maeder DL, Jagus R. Phylogenetic analysis of eIF4E-family members. BMC Evol Biol. 2005;5:48. doi: 10.1186/1471-2148-5-48. PubMed DOI PMC

Rom E, Kim HC, Gingras AC, Marcotrigiano J, Favre D, Olsen H, Burley SK, Sonenberg N. Cloning and characterization of 4EHP, a novel mammalian eIF4E-related cap-binding protein. J Biol Chem. 1998;273(21):13104–13109. doi: 10.1074/jbc.273.21.13104. PubMed DOI

Joshi B, Cameron A, Jagus R. Characterization of mammalian eIF4E-family members. Eur J Biochem. 2004;271(11):2189–2203. doi: 10.1111/j.1432-1033.2004.04149.x. PubMed DOI

Osborne MJ, Volpon L, Kornblatt JA, Culjkovic-Kraljacic B, Baguet A, Borden KL. eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition. Proc Natl Acad Sci USA. 2013;110(10):3877–3882. doi: 10.1073/pnas.1216862110. PubMed DOI PMC

Cho PF, Poulin F, Cho-Park YA, Cho-Park IB, Chicoine JD, Lasko P, Sonenberg N. A new paradigm for translational control: inhibition via 5′–3′ mRNA tethering by Bicoid and the eIF4E cognate 4EHP. Cell. 2005;121(3):411–423. doi: 10.1016/j.cell.2005.02.024. PubMed DOI

Cho PF, Gamberi C, Cho-Park YA, Cho-Park IB, Lasko P, Sonenberg N. Cap-dependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos. Curr Biol. 2006;16(20):2035–2041. doi: 10.1016/j.cub.2006.08.093. PubMed DOI PMC

Villaescusa JC, Buratti C, Penkov D, Mathiasen L, Planaguma J, Ferretti E, Blasi F. Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation. PLoS ONE. 2009;4(4):e5213. doi: 10.1371/journal.pone.0005213. PubMed DOI PMC

Tao X, Gao G. Tristetraprolin recruits eukaryotic initiation factor 4E2 to repress translation of AU-rich element-containing mRNAs. Mol Cell Biol. 2015;35(22):3921–3932. doi: 10.1128/MCB.00845-15. PubMed DOI PMC

Uniacke J, Holterman CE, Lachance G, Franovic A, Jacob MD, Fabian MR, Payette J, Holcik M, Pause A, Lee S. An oxygen-regulated switch in the protein synthesis machinery. Nature. 2012;486(7401):126–129. PubMed PMC

Ho JJ, Wang M, Audas TE, Kwon D, Carlsson SK, Timpano S, Evagelou SL, Brothers S, Gonzalgo ML, Krieger JR, et al. Systemic reprogramming of translation efficiencies on oxygen stimulus. Cell reports. 2016;14(6):1293–1300. doi: 10.1016/j.celrep.2016.01.036. PubMed DOI PMC

Dinkova TD, Keiper BD, Korneeva NL, Aamodt EJ, Rhoads RE. Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform. Mol Cell Biol. 2005;25(1):100–113. doi: 10.1128/MCB.25.1.100-113.2005. PubMed DOI PMC

Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in primary solid tumors. Nat Genet. 2003;33(1):49–54. doi: 10.1038/ng1060. PubMed DOI

Landon AL, Muniandy PA, Shetty AC, Lehrmann E, Volpon L, Houng S, Zhang Y, Dai B, Peroutka R, Mazan-Mamczarz K, et al. MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL. Nat Commun. 2014;5:5413. doi: 10.1038/ncomms6413. PubMed DOI PMC

Anderson P, Kedersha N. RNA granules. J Cell Biol. 2006;172(6):803–808. doi: 10.1083/jcb.200512082. PubMed DOI PMC

Andrei MA, Ingelfinger D, Heintzmann R, Achsel T, Rivera-Pomar R, Luhrmann R. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA. 2005;11(5):717–727. doi: 10.1261/rna.2340405. PubMed DOI PMC

Suzuki Y, Minami M, Suzuki M, Abe K, Zenno S, Tsujimoto M, Matsumoto K, Minami Y. The Hsp90 inhibitor geldanamycin abrogates colocalization of eIF4E and eIF4E-transporter into stress granules and association of eIF4E with eIF4G. J Biol Chem. 2009;284(51):35597–35604. doi: 10.1074/jbc.M109.036285. PubMed DOI PMC

Ferraiuolo MA, Basak S, Dostie J, Murray EL, Schoenberg DR, Sonenberg N. A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J Cell Biol. 2005;170(6):913–924. doi: 10.1083/jcb.200504039. PubMed DOI PMC

Fournier MJ, Coudert L, Mellaoui S, Adjibade P, Gareau C, Cote MF, Sonenberg N, Gaudreault RC, Mazroui R. Inactivation of the mTORC1-eukaryotic translation initiation factor 4E pathway alters stress granule formation. Mol Cell Biol. 2013;33(11):2285–2301. doi: 10.1128/MCB.01517-12. PubMed DOI PMC

Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol. 2001;21(23):7971–7980. doi: 10.1128/MCB.21.23.7971-7980.2001. PubMed DOI PMC

McEwen E, Kedersha N, Song B, Scheuner D, Gilks N, Han A, Chen JJ, Anderson P, Kaufman RJ. Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure. J Biol Chem. 2005;280(17):16925–16933. doi: 10.1074/jbc.M412882200. PubMed DOI

Mokas S, Mills JR, Garreau C, Fournier MJ, Robert F, Arya P, Kaufman RJ, Pelletier J, Mazroui R. Uncoupling stress granule assembly and translation initiation inhibition. Mol Biol Cell. 2009;20(11):2673–2683. doi: 10.1091/mbc.E08-10-1061. PubMed DOI PMC

Dang Y, Kedersha N, Low WK, Romo D, Gorospe M, Kaufman R, Anderson P, Liu JO. Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A. J Biol Chem. 2006;281(43):32870–32878. doi: 10.1074/jbc.M606149200. PubMed DOI

Fujimura K, Sasaki AT, Anderson P. Selenite targets eIF4E-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules. Nucleic Acids Res. 2012;40(16):8099–8110. doi: 10.1093/nar/gks566. PubMed DOI PMC

Kedersha N, Tisdale S, Hickman T, Anderson P. Real-time and quantitative imaging of mammalian stress granules and processing bodies. Methods Enzymol. 2008;448:521–552. doi: 10.1016/S0076-6879(08)02626-8. PubMed DOI

Stoecklin G, Kedersha N. Relationship of GW/P-bodies with stress granules. Adv Exp Med Biol. 2013;768:197–211. doi: 10.1007/978-1-4614-5107-5_12. PubMed DOI PMC

Eulalio A, Behm-Ansmant I, Izaurralde E. P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Bio. 2007;8(1):9–22. doi: 10.1038/nrm2080. PubMed DOI

Ayache J, Benard M, Ernoult-Lange M, Minshall N, Standart N, Kress M, Weil D. P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes. Mol Biol Cell. 2015;26(14):2579–2595. doi: 10.1091/mbc.E15-03-0136. PubMed DOI PMC

Anderson P, Kedersha N. RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol. 2009;10(6):430–436. doi: 10.1038/nrm2694. PubMed DOI

Lavut A, Raveh D. Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability. PLoS Genet. 2012;8(2):e1002527. doi: 10.1371/journal.pgen.1002527. PubMed DOI PMC

Kedersha N, Stoecklin G, Ayodele M, Yacono P, Lykke-Andersen J, Fritzler MJ, Scheuner D, Kaufman RJ, Golan DE, Anderson P. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol. 2005;169(6):871–884. doi: 10.1083/jcb.200502088. PubMed DOI PMC

Rong L, Livingstone M, Sukarieh R, Petroulakis E, Gingras AC, Crosby K, Smith B, Polakiewicz RD, Pelletier J, Ferraiuolo MA, et al. Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs. RNA. 2008;14(7):1318–1327. doi: 10.1261/rna.950608. PubMed DOI PMC

Sukarieh R, Sonenberg N, Pelletier J. The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response. Am J Physiol Cell Physiol. 2009;296(5):C1207–C1217. doi: 10.1152/ajpcell.00511.2008. PubMed DOI PMC

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162(1):156–159. doi: 10.1016/0003-2697(87)90021-2. PubMed DOI

Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1(6):2856–2860. doi: 10.1038/nprot.2006.468. PubMed DOI

Zamostna B, Novak J, Vopalensky V, Masek T, Burysek L, Pospisek M. N-terminal domain of nuclear IL-1alpha shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex. PLoS ONE. 2012;7(8):e41801. doi: 10.1371/journal.pone.0041801. PubMed DOI PMC

Masek T, Valasek L, Pospisek M. Polysome analysis and RNA purification from sucrose gradients. Methods Mol Biol. 2011;703:293–309. doi: 10.1007/978-1-59745-248-9_20. PubMed DOI

Tomoo K, Matsushita Y, Fujisaki H, Abiko F, Shen X, Taniguchi T, Miyagawa H, Kitamura K, Miura K, Ishida T. Structural basis for mRNA cap-binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods. Biochim Biophys Acta. 2005;1753(2):191–208. doi: 10.1016/j.bbapap.2005.07.023. PubMed DOI

Volpon L, Osborne MJ, Topisirovic I, Siddiqui N, Borden KL. Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands. EMBO J. 2006;25(21):5138–5149. doi: 10.1038/sj.emboj.7601380. PubMed DOI PMC

Brown CJ, McNae I, Fischer PM, Walkinshaw MD. Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives. J Mol Biol. 2007;372(1):7–15. doi: 10.1016/j.jmb.2007.06.033. PubMed DOI

Fukuyo A, In Y, Ishida T, Tomoo K. Structural scaffold for eIF4E binding selectivity of 4E-BP isoforms: crystal structure of eIF4E binding region of 4E-BP2 and its comparison with that of 4E-BP1. J Pept Sci. 2011;17(9):650–657. doi: 10.1002/psc.1384. PubMed DOI

Chu CY, Rana TM. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol. 2006;4(7):e210. doi: 10.1371/journal.pbio.0040210. PubMed DOI PMC

Wilczynska A, Aigueperse C, Kress M, Dautry F, Weil D. The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules. J Cell Sci. 2005;118(5):981–992. doi: 10.1242/jcs.01692. PubMed DOI

Souquere S, Mollet S, Kress M, Dautry F, Pierron G, Weil D. Unravelling the ultrastructure of stress granules and associated P-bodies in human cells. J Cell Sci. 2009;122(Pt 20):3619–3626. doi: 10.1242/jcs.054437. PubMed DOI

Wang ET, Sandberg R, Luo SJ, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB. Alternative isoform regulation in human tissue transcriptomes. Nature. 2008;456(7221):470–476. doi: 10.1038/nature07509. PubMed DOI PMC

Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40(12):1413–1415. doi: 10.1038/ng.259. PubMed DOI

Scheper GC, Parra JL, Wilson M, Van Kollenburg B, Vertegaal AC, Han ZG, Proud CG. The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization. Mol Cell Biol. 2003;23(16):5692–5705. doi: 10.1128/MCB.23.16.5692-5705.2003. PubMed DOI PMC

Kubacka D, Kamenska A, Broomhead H, Minshall N, Darzynkiewicz E, Standart N. Investigating the consequences of eIF4E2 (4EHP) interaction with 4E-transporter on its cellular distribution in HeLa cells. PLoS ONE. 2013;8(8):e72761. doi: 10.1371/journal.pone.0072761. PubMed DOI PMC

Tcherkezian J, Brittis PA, Thomas F, Roux PP, Flanagan JG. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell. 2010;141(4):632–644. doi: 10.1016/j.cell.2010.04.008. PubMed DOI PMC

Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson A, Kampf C, Sjostedt E, Asplund A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347(6220):1260419. doi: 10.1126/science.1260419. PubMed DOI

Mollet S, Cougot N, Wilczynska A, Dautry F, Kress M, Bertrand E, Weil D. Translationally repressed mRNA transiently cycles through stress granules during stress. Mol Biol Cell. 2008;19(10):4469–4479. doi: 10.1091/mbc.E08-05-0499. PubMed DOI PMC

Kong J, Lasko P. Translational control in cellular and developmental processes. Nat Rev Genet. 2012;13(6):383–394. doi: 10.1038/nrg3184. PubMed DOI

Osborne MJ, Borden KL. The eukaryotic translation initiation factor eIF4E in the nucleus: taking the road less traveled. Immunol Rev. 2015;263(1):210–223. doi: 10.1111/imr.12240. PubMed DOI PMC

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method

Major splice variants and multiple polyadenylation site utilization in mRNAs encoding human translation initiation factors eIF4E1 and eIF4E3 regulate the translational regulators?