Tethered Function Assays to Elucidate the Role of RNA-Binding Proteins

Abstract

The fate of each RNA molecule is strongly determined by RNA-binding proteins (RBPs) which accompany transcripts from its synthesis to its degradation. To elucidate the effect of a specific RBP on bound RNA, it can be artificially recruited to a specific site on a reporter mRNA that can be followed by a variety of methods. In this so-called tethering assay, the protein of interest (POI) is fused to the coat protein of the MS2 bacteriophage and expressed in your favorite cells together with a reporter gene containing MS2 binding sites. The MS2 binding sites are recognized by the MS2 coat protein (MS2CP) with high affinity and specificity and by doing so, the POI is tethered to the reporter RNA. Here, we describe how with the help of this assay the human cytoplasmic poly(A) binding protein is recruited to a mini-μ RNA reporter, thereby influencing the stability of the reporter transcript.

Keywords: MS2 binding sites; MS2 coat protein (MS2CP); Nonsense-mediated mRNA decay (NMD); Poly(A) binding protein; RNA-binding protein; Tethered protein; Tethering assay.