Protocol

Analysis of Nonsense-Mediated mRNA Decay by Monitoring mRNA Half-Lives in Mammalian Cells

¹ Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
² Advanced Medical Research Center, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
³ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

⁴Corresponding authors (ohnos{at}med.yokohama-cu.ac.jp; yamasita{at}yokohama-cu.ac.jp).

INTRODUCTION

This protocol uses a tetracycline-response-element-regulated (Tet-Off) promoter system and Northern blotting to analyze the half-lives of premature termination codon (PTC)-containing mRNAs following the activation or suppression of the nonsense-mediated mRNA decay (NMD) pathway in mammalian cells. Reporter plasmids are cotransfected with or without effector plasmid(s) and/or small interfering RNA(s) (siRNA[s]) targeted to genes suspected of involvement in NMD. After transfection, cells are harvested and reseeded to five or six plates. Thirty-six to 72 h after transfection, the cells are treated with doxycycline (or tetracycline) to terminate reporter mRNA transcription from the Tet-Off promoter. Cells are then harvested at specific time points, and RNAs are purified for Northern blot analysis. Although various alternative methods (e.g., RNase protection assay, reverse transcriptase polymerase chain reaction [RT-PCR], and quantitative-RT-PCR [qRT-PCR]) are available, the use of Northern blotting to analyze mRNA half-lives to confirm correct mRNA processing is highly recommended. Using this protocol to analyze mRNA half-lives provides an assay that is dependent on the activities of NMD transacting factor(s) in mammalian cells.