Defining nonsense-mediated mRNA decay intermediates in human cells

Abstract Nonsense-mediated mRNA decay (NMD) is a cellular mRNA degradation mechanism that inhibits the expression of aberrant mRNAs harboring premature termination codons (PTCs). Recent progress in transcriptome-wide sequencing techniques has revealed that NMD also degrades approximately 5–30% of non-mutated cellular mRNAs in a way that can be regulated in response to various cellular signals. In mammals, NMD is governed by the central NMD factor UPF1, which is activated by phosphorylation after translation terminates at a nonsense codon that triggers NMD. We have found that immunoprecipitation using an antibody that is specific for phosphorylated UPF1 is a useful tool to define not only cellular NMD targets but also the nature of NMD decay intermediates and, thus, the process of NMD. To this end, we describe here a detailed protocol for what we call “NMD degradome sequencing” using high-throughput technology.