title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.
abstract: Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Castelli
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Talavera
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kershaw
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Mohammad-Qureshi
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Costello
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Rowe
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; Manchester Institute of Biotechnology (MIB), The University of Manchester, Manchester, United Kingdom.
authors.forename: Paul F G
authors.initials: PF
authors.lastname: Sims
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Grant
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Hubbard
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Ashe
authors.affiliation: The Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D015536:Down-Regulation; D063185:Epigenetic Repression; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D011485:Protein Binding; D014176:Protein Biosynthesis; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C555411:Caf20 protein, S cerevisiae; C064034:ERS1 protein, S cerevisiae; C555412:Eap1 protein, S cerevisiae; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D008565:Membrane Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors