eIF3 targets cell-proliferation messenger RNAs for translational activation or repression

Abstract

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.

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Figure 1: PAR-CLIP of the multi-protein translation initiation factor complex eIF3.
Figure 2: Analysis and validation of eIF3 PAR-CLIP-derived binding sites.
Figure 3: eIF3 is a positive and negative transcript-specific translational regulator.
Figure 4: Opposing translation phenotypes are driven by different modes of eIF3–mRNA binding.

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Primary accessions

Gene Expression Omnibus

Data deposits

All data have been deposited in the Gene Expression Omnibus under accession number GSE65004.

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Acknowledgements

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.

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Contributions

Experiments were designed by A.S.Y.L. in consultation with J.H.D.C. All experiments and analyses were performed by A.S.Y.L. P.J.K. performed gel shift assays and assisted with biochemistry. The manuscript was written by A.S.Y.L. and J.H.D.C. All authors contributed to editing the manuscript and support the conclusions.

Corresponding author

Correspondence to Jamie H. D. Cate.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 PAR-CLIP reveals eIF3a, b, d and g bind to RNA.

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.

Extended Data Figure 2 Analysis of eIF3 PAR-CLIP targets.

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.

Extended Data Figure 3 Conservation of c-JUN and BTG1 eIF3-binding sites in primates and mammals.

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.

Extended Data Figure 5 Luciferase activity of c-JUN and BTG1 mutants in cells. a,

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.

Extended Data Figure 7 Schematic of eIF3 subunit localization on the small ribosomal subunit.

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.

Supplementary information

Supplementary Table 1

This file contains the data for Consensus PAR-CLIP eIF3–RNA clusters. (XLS 268 kb)

Supplementary Table 2

This file contains the data for FPKM for eIF3-crosslinked mRNAs. (XLS 160 kb)

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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

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