Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display

Abstract

Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification of mRNAs associated with a protein. Methods are detailed for expressing and purifying fusion proteins that are used to isolate substrate mRNPs employing differential display technology. Lastly, experiments are described to confirm that the RNAs identified are indeed bonafide substrates for an RNA-binding protein. As the number of known RNA-binding proteins increases, of which many are involved in genetic disorders, it is essential that methodologies exist to identify RNA-protein interactions to better understand the manifestation of disease.