title: Proteomic Analysis Reveals Branch-specific Regulation of the Unfolded Protein Response by Nonsense-mediated mRNA Decay.
abstract: Nonsense-mediated mRNA decay (NMD) has originally been described as a surveillance mechanism to inhibit the expression of mRNAs with truncated open reading frames (ORFs) and to contribute to the fidelity of gene expression. It is now recognized that NMD also controls the expression of physiological genes with "intact" mRNA. Stress can decrease NMD efficiency and thus increase the mRNA levels of physiological NMD targets. As stress can also inhibit translation, the net outcome for shaping the proteome is difficult to predict. We have thus analyzed de novo protein synthesis in response to NMD inhibition or the induction of mild endoplasmic reticulum (ER) stress by treatment of cells with the reducing agent dithiotreitol (DTT). For this purpose, we combined pulsed azidohomoalanine (AHA) and stable isotope labeling by amino acids in cell culture (SILAC). Labeled proteins were purified by click chemistry-based covalent coupling to agarose beads, trypsinized, fractionated, and analyzed by mass spectrometry (MS). We find that mild ER stress up-regulates the de novo synthesis of components of all three branches of the unfolded protein response (PERK, IRE1 and ATF6) without increasing eIF2α phosphorylation or impairing of protein translation. In contrast, inhibition of NMD induces de novo protein synthesis of downstream targets of the PERK and IRE1 pathways, whereas we could not detect regulation of ATF6-responsive genes. These data thus support a model that implicates a positive feedback loop of ER stress inhibiting NMD efficiency which further promotes the ER stress response in a branch-specific manner.
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; §Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany;
authors.forename: Jana
authors.initials: J
authors.lastname: Sieber
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; §Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany; ¶European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hauer
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; §Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany;
authors.forename: Madhuri
authors.initials: M
authors.lastname: Bhuvanagiri
authors.affiliation: ¶European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Leicht
authors.affiliation: ¶European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; §Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany;
authors.forename: Gabriele
authors.initials: G
authors.lastname: Neu-Yilik
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; ¶European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Matthias W
authors.identifier: 0000-0002-4023-7876
authors.initials: MW
authors.lastname: Hentze
authors.affiliation: From the ‡Molecular Medicine Partnership Unit, European Molecular Biology Laboratory, University of Heidelberg, Heidelberg, Germany; §Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany; andreas.kulozik@med.uni-heidelberg.de.
authors.forename: Andreas E
authors.initials: AE
authors.lastname: Kulozik
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology*; D059865:Endoplasmic Reticulum Stress; D053263:Gene Regulatory Networks / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D059625:Liquid-Liquid Extraction; D059365:Nonsense Mediated mRNA Decay / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D056811:Unfolded Protein Response / Q000187:drug effects*
chemical_list: C506098:azidohomoalanine; D000409:Alanine; D004229:Dithiothreitol