Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis and stress responses1,2. The 13-subunit, 800-kilodalton eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation3. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects4. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP)5. eIF3 binds to a highly specific program of messenger RNAs involved in cell growth control processes, including cell cycling, differentiation and apoptosis, via the mRNA 5′ untranslated region. Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding the cell proliferation regulators c-JUN and BTG1 reveals that eIF3 uses different modes of RNA stem–loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Stumpf, C. R. & Ruggero, D. The cancerous translation apparatus. Curr. Opin. Genet. Dev. 21, 474–483 (2011)
Silvera, D., Formenti, S. C. & Schneider, R. J. Translational control in cancer. Nature Rev. Cancer 10, 254–266 (2010)
Jackson, R. J., Hellen, C. U. & Pestova, T. V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Rev. Mol. Cell Biol. 11, 113–127 (2010)
Hershey, J. W. Regulation of protein synthesis and the role of eIF3 in cancer. Braz. J. Med. Biol. Res. 43, 920–930 (2010)
Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010)
Choudhuri, A., Maitra, U. & Evans, T. Translation initiation factor eIF3h targets specific transcripts to polysomes during embryogenesis. Proc. Natl Acad. Sci. USA 110, 9818–9823 (2013)
Curran, S. P. & Ruvkun, G. Lifespan regulation by evolutionarily conserved genes essential for viability. PLoS Genet. 3, e56 (2007)
Zhang, L., Pan, X. & Hershey, J. W. Individual overexpression of five subunits of human translation initiation factor eIF3 promotes malignant transformation of immortal fibroblast cells. J. Biol. Chem. 282, 5790–5800 (2007)
Fraser, C. S. & Doudna, J. A. Structural and mechanistic insights into hepatitis C viral translation initiation. Nature Rev. Microbiol. 5, 29–38 (2007)
Corcoran, D. L. et al. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011)
Pisarev, A. V., Kolupaeva, V. G., Yusupov, M. M., Hellen, C. U. & Pestova, T. V. Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes. EMBO J. 27, 1609–1621 (2008)
Hashem, Y. et al. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153, 1108–1119 (2013)
Hashem, Y. et al. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503, 539–543 (2013)
Erzberger, J. P. et al. Molecular architecture of the 40SeIF1eIF3 translation initiation complex. Cell 158, 1123–1135 (2014)
Wisdom, R., Johnson, R. S. & Moore, C. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 18, 188–197 (1999)
Rouault, J. P. et al. BTG1, a member of a new family of antiproliferative genes. EMBO J. 11, 1663–1670 (1992)
Bakker, W. J. et al. FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1. J. Cell Biol. 164, 175–184 (2004)
Blau, L. et al. Aberrant expression of c-Jun in glioblastoma by internal ribosome entry site (IRES)-mediated translational activation. Proc. Natl Acad. Sci. USA 109, E2875–E2884 (2012)
Kozak, M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol. Cell. Biol. 9, 5134–5142 (1989)
Johnson, R., Spiegelman, B., Hanahan, D. & Wisdom, R. Cellular transformation and malignancy induced by ras require c-jun. Mol. Cell. Biol. 16, 4504–4511 (1996)
Waanders, E. et al. The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution. PLoS Genet. 8, e1002533 (2012)
Pincheira, R., Chen, Q. & Zhang, J. T. Identification of a 170-kDa protein over-expressed in lung cancers. Br. J. Cancer 84, 1520–1527 (2001)
Carlsten, J. O., Zhu, X. & Gustafsson, C. M. The multitalented Mediator complex. Trends Biochem. Sci. 38, 531–537 (2013)
Andaya, A., Villa, N., Jia, W., Fraser, C. S. & Leary, J. A. Phosphorylation stoichiometries of human eukaryotic initiation factors. Int. J. Mol. Sci. 15, 11523–11538 (2014)
Damoc, E. et al. Structural characterization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry. Mol. Cell. Proteomics 6, 1135–1146 (2007)
Holz, M. K., Ballif, B. A., Gygi, S. P. & Blenis, J. mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 123, 569–580 (2005)
Kondrashov, N. et al. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383–397 (2011)
Lee, A. S., Burdeinick-Kerr, R. & Whelan, S. P. A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc. Natl Acad. Sci. USA 110, 324–329 (2013)
Ule, J., Jensen, K., Mele, A. & Darnell, R. B. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005)
Kranzusch, P. J. et al. Structure-guided reprogramming of human cGAS dinucleotide linkage specificity. Cell 158, 1011–1021 (2014)
Rakotondrafara, A. M. & Hentze, M. W. An efficient factor-depleted mammalian in vitro translation system. Nature Protocols 6, 563–571 (2011)
Sun, C. et al. Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3). Proc. Natl Acad. Sci. USA 108, 20473–20478 (2011)
Davidovich, C., Zheng, L., Goodrich, K. J. & Cech, T. R. Promiscuous RNA binding by Polycomb repressive complex 2. Nature Struct. Mol. Biol. 20, 1250–1257 (2013)
Kranzusch, P. J. et al. Assembly of a functional Machupo virus polymerase complex. Proc. Natl Acad. Sci. USA 107, 20069–20074 (2010)
Bai, Y., Zhou, K. & Doudna, J. A. Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery. Nucleic Acids Res. 41, 7861–7874 (2013)
Vasa, S. M., Guex, N., Wilkinson, K. A., Weeks, K. M. & Giddings, M. C. ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA 14, 1979–1990 (2008)
Hafner, M. et al. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods 58, 164–170 (2012)
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856–2860 (2007)
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)
Lebedeva, S. et al. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol. Cell 43, 340–352 (2011)
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)
The authors thank J. Doudna, D. Ruggero, D. Black, M. Truitt, A. Tambe, Y. Bai and K. Chat for discussions. HeLa cytoplasm was a gift from J. Fang. This work used the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley, supported by National Institutes of Health (NIH) S10 Instrumentation Grants S10RR029668 and S10RR027303; and the Vincent J. Proteomics/Mass Spectrometry Laboratory at University of California, Berkeley, supported in part by NIH S10 Instrumentation Grant S10RR025622. This work was funded by the National Institute of General Medical Sciences Center for RNA Systems Biology (A.S.Y.L. and J.H.D.C.). A.S.Y.L is supported as an American Cancer Society Postdoctoral Fellow (PF-14-108-01-RMC) and P.J.K. is supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.
The authors declare no competing financial interests.
Extended data figures and tables
a, Mass spectrometry identification of trypsin-released peptides from RNA-crosslinked eIF3 subunits. Peptides identified by mass spectrometry are highlighted in pink. b, c, Crosslinking and denaturing immunoprecipitation to validate subunit identification. As eIF3d and g co-migrate with eIF3l and e/f, respectively, subunit identification was validated by immunoprecipitation of individual proteins after crosslinking and treatment of lysates with SDS treatment and boiling.
a, Scatterplot of fragments per kilobase of exon per million reads (FPKM) of all mRNAs expressed in 293T cells. mRNAs that are eIF3 PAR-CLIP targets are highlighted in red. b, Scatterplot of correlation between mRNA expression and PAR-CLIP read coverage for mRNAs that are eIF3 PAR-CLIP targets. The simple linear regression line is plotted in blue, with the 95% confidence region shaded in grey.
a, b, The eIF3-binding site is indicated in cyan. nt, nucleotides. a, c-JUN GenBank accessions are: human (NM_002228.3, Homo sapiens), chimpanzee (XM_513442.5, Pan troglodytes), gorilla (XM_004025880.1, Gorilla gorilla), orangutan (XM_002810763.3, Pongo abelii), rhesus macaque (NM_001265850.2, Macaca mulatta), marmoset (XM_002750880.3, Callithrix jacchus), mouse (NM_010591.2, Mus musculus), cat (XM_006934825.1, Felis catus). b, BTG1 GenBank accessions are: human (NM_001731.2, Homo sapiens), chimpanzee (XM_509262.3, Pan troglodytes), orangutan (XM_002823578.2, Pongo abelii), rhesus macaque (NM_001266672.1, Macaca mulatta), marmoset (XM_002752814.3, Callithrix jacchus), mouse (NM_007569.2, Mus musculus), cat (XM_006933950.1, Felis catus), cow (NM_173999.3, Bos taurus).
Extended Data Figure 4 Interactions between native and recombinant eIF3 and the c-JUN and BTG1 RNA stem–loops.
a, Coomassie blue staining of purified native HeLa eIF3 or recombinant eIF3, resolved by SDS–PAGE. b, Representative native agarose gel electrophoresis shows a specific and binary interaction between native (Nat) and recombinant (Rec) eIF3 and the wild-type (WT) c-JUN stem–loop structure, but not with the mutated stem–loop or the wild-type BTG1 stem–loop.
b, Luciferase activity in 293T cells transfected with mRNAs containing the c-JUN 5′ UTR with a mutated stem–loop (a) or the PSMB6 5′ UTR-BTG1 stem–loop chimaera (b). Mut, mutant; Rev, transversed; SL, stem–loop; WT, wild type. The results are given as the mean ± s.d. of three independent experiments, each performed in triplicate.
Extended Data Figure 6 Bypassing eIF3 translational control in H1299 cells reduces cell invasiveness.
a, Functional classification of eIF3-bound RNAs. b, Representative western blot analysis of eIF3a expression levels in H1299 and IMR90 cells. GAPDH was detected as a loading control for normalized protein levels. c, Representative image of Matrigel invasion by H1299 or IMR90 cells. d, BTG1 protein levels after overexpression in H1299 cells. HSP90 was detected as a loading control. e, Matrigel invasion assay in H1299 cells after overexpression of BTG1. ORF, open reading frame. f, c-JUN protein levels after siRNA-mediated knockdown in H1299 cells. NT, non-targeting. g, Matrigel invasion assay in H1299 cells after knockdown of c-JUN. The results of e and g are given as the mean ± s.d. of three independent experiments, each performed in duplicate.
The eIF3 subunits bound to RNA in the PAR-CLIP experiment, eIF3a, b and g, form a nexus in the distal eIF3 region. The location of eIF3d has not been assigned, and the schematic is adapted from ref. 14.
About this article
Cite this article
Lee, A., Kranzusch, P. & Cate, J. eIF3 targets cell-proliferation messenger RNAs for translational activation or repression. Nature 522, 111–114 (2015). https://doi.org/10.1038/nature14267
EIF3H Knockdown Inhibits Malignant Melanoma through Regulating Cell Proliferation, Apoptosis and Cell Cycle
Experimental Cell Research (2021)
Trends in Genetics (2021)
European Journal of Immunology (2021)
Berberine induces anti-atopic dermatitis effects through the downregulation of cutaneous EIF3F and MALT1 in NC/Nga mice with atopy-like dermatitis
Biochemical Pharmacology (2021)
Molecular Cell (2021)