title: The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA.
abstract: DNA damage, stalled replication forks, errors in mRNA splicing, and availability of nutrients activate specific phosphatidylinositiol-3 kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2, or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double-strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.
authors.affiliation: Department of Biochemistry and Biophysics, The University of Rochester, Rochester, NY, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Gewandter
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Michael A
authors.initials: MA
authors.lastname: O'Reilly
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D017346:Protein Serine-Threonine Kinases; D020365:RNA Helicases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C468286:UPF2 protein, human; C576324:ATM protein, human; C514704:SMG1 protein, human; C101828:UPF1 protein, human