A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay

Author:

Kolakada Divya12,Campbell Amy E2,Galvis Laura Baquero2,Li Zhongyou2,Lore Mlana12,Jagannathan Sujatha23ORCID

Affiliation:

1. Molecular Biology Graduate Program, University of Colorado Anschutz Medical Campus , Aurora , CO  80045 , USA

2. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus , Aurora , CO  80045 , USA

3. RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus , Aurora , CO  80045 , USA

Abstract

Abstract Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

Funder

University of Colorado Anschutz Medical Campus

National Institutes of Health

American Heart Association Pre-Doctoral Award

Cystic Fibrosis Foundation Postdoctoral Research Fellowship Award

University of Colorado Cancer Center

Publisher

Oxford University Press (OUP)

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