Dynamic regulation of RNA molecules is critical to the survival and development of cells. Messenger RNAs are transcribed in the nucleus as intron-containing pre-mRNAs and bound by RNA-binding proteins, which control their fate by regulating RNA stability, splicing, polyadenylation, translation, and cellular localization. Most RBPs have distinct mRNA-binding and functional domains; thus, the function of an RBP can be studied independently of RNA-binding by artificially recruiting the RBP to a reporter RNA and then measuring the effect of RBP recruitment on reporter splicing, stability, translational efficiency, or intracellular trafficking. These tethered function assays therefore do not require prior knowledge of the RBP's endogenous RNA targets or its binding sites within these RNAs. Here, we provide an overview of the experimental strategy and the strengths and limitations of common tethering systems. We illustrate specific examples of the application of the assay in elucidating the function of various classes of RBPs. We also discuss how classic tethering assay approaches and insights gained from them have been empowered by more recent technological advances, including efficient genome editing and high-throughput RNA-sequencing.
Keywords: BoxB; Lambda N; MS2 coat protein; Nucleocytoplasmic transport; RNA stability; Subcellular localization; Tethered function assays; Translation.