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Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy

Lookup NU author(s): Dr Anze Zupanic, Dr Catherine Meplan, Grazielle Huguenin, Professor John Edward Hesketh, Dr Daryl Shanley

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


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.


Publication metadata

Author(s): Zupanic A, Meplan C, Huguenin GVB, Hesketh JE, Shanley DP

Publication type: Article

Publication status: Published

Journal: RNA

Year: 2016

Volume: 22

Issue: 7

Pages: 1076-1084

Print publication date: 01/07/2016

Online publication date: 20/05/2016

Acceptance date: 02/04/2016

Date deposited: 12/08/2016

ISSN (print): 1355-8382

ISSN (electronic): 1469-9001

Publisher: Cold Spring Harbor Laboratory Press

URL: http://dx.doi.org/10.1261/rna.055749.115

DOI: 10.1261/rna.055749.115


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