title: Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.
abstract: The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism.
authors.affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Newcastle University, Newcastle-upon-Tyne NE4 5PL, United Kingdom Eawag, Institute for Aquatic Science and Technology, 8600 Dübendorf, Switzerland.
authors.forename: Anze
authors.identifier: 0000-0003-3303-9086
authors.initials: A
authors.lastname: Zupanic
authors.affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Newcastle University, Newcastle-upon-Tyne NE4 5PL, United Kingdom Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom.
authors.forename: Catherine
authors.initials: C
authors.lastname: Meplan
authors.affiliation: Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom Faculty of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, CEP: 21941-902, Brazil.
authors.forename: Grazielle V B
authors.initials: GV
authors.lastname: Huguenin
authors.affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Newcastle University, Newcastle-upon-Tyne NE4 5PL, United Kingdom Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom.
authors.forename: John E
authors.initials: JE
authors.lastname: Hesketh
authors.affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Newcastle University, Newcastle-upon-Tyne NE4 5PL, United Kingdom Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom.
authors.forename: Daryl P
authors.initials: DP
authors.lastname: Shanley
mesh_terms: D018938:Caco-2 Cells; D055785:Gene Knockdown Techniques; D006801:Humans; D008962:Models, Theoretical; D059365:Nonsense Mediated mRNA Decay; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051140:Selenoproteins / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D051140:Selenoproteins
keywords: mathematical modeling; nonsense-mediated decay; premature termination; selenoprotein; translation