RNA remodeling by RNA chaperones monitored by RNA structure probing

Abstract

RNA structure probing enables the characterization of RNA secondary structures by established procedures such as the enzyme- or chemical-based detection of single- or double-stranded regions. A specific type of application involves the detection of changes of RNA structures and conformations that are induced by proteins with RNA chaperone activity. This chapter outlines a protocol to analyze RNA structures in vitro in the presence of an RNA-binding protein with RNA chaperone activity. For this purpose, we make use of the methylating agents dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT). DMS and CMCT specifically modify nucleotides that are not involved in base-pairing or tertiary structure hydrogen bonding and that are not protected by a ligand such as a protein. Modified bases are identified by primer extension. As an example, we describe how the RNA chaperone activity of an isoform of the RNA-binding protein AUF1 induces the flaviviral RNA switch required for viral genome cyclization and viral replication. This chapter includes comprehensive protocols for in vitro synthesis of RNA, 32P-5′-end labeling of DNA primers, primer extension, as well as the preparation and running of analytical gels. The described methodology should be applicable to any other RNA and protein of interest to identify protein-directed RNA remodeling.