A tethering approach to study proteins that activate mRNA turnover in human cells.

The regulation of mRNA turnover occurs in part through the action of mRNA-binding proteins that recognize specific nucleotide sequences and either activate or inhibit the decay of transcripts to which they are bound. In many cases, multiple mRNA-binding proteins, including those with opposing functions, bind to the same RNA sequence. This can make the study of the function of any one of these proteins difficult. Furthermore, monitoring endogenous mRNA decay rates using drugs that inhibit transcription (e.g., actinomycin D) can introduce pleiotropic effects. One way to circumvent these problems is to tether the protein of interest (POI) through a heterologous RNA-binding domain to an inducible reporter mRNA and measure the effect of the bound protein on mRNA decay. In this chapter, we illustrate the use of the tethering technique to study the role of a particular mRNA-binding protein, TTP, on the decay of an otherwise stable mRNA to which it is tethered through a fusion to the bacteriophage MS2 coat protein.