Tethering assays to investigate nonsense-mediated mRNA decay activating proteins

Nonsense-mediated mRNA decay (NMD) is activated by exon-junction complexes (EJCs) that are located downstream of the termination codon of the substrate mRNAs. This situation can be imitated by tethering components of the EJC to the 3' untranslated region (3' UTR) of a reporter mRNA. Here we describe the detailed use of two analogous tethering systems that are based on the coat protein of bacteriophage MS2 or on the 22 amino acid RNA-binding domain of the bacteriophage lambda-antiterminator protein N (lambdaN-peptide). These polypeptides are fused as tags to proteins of interest. Their respective RNA binding sites are inserted into reporter mRNAs. This enables recruitment of the NMD activity of the fusion protein to an NMD-activating position, bypassing the requirement for splicing. In this chapter we explicate the cloning of appropriate reporter plasmids and the setup of a tethering experiment with the necessary control experiments. Advantages of the different systems and tags are discussed.