Selective Isolation of TOP3B•mRNA Covalent Intermediates Using Denaturing Oligo-dT Pulldown.

The deletion and mutation of Topoisomerase 3β (TOP3B) is linked to multiple neurological disorders and is the only known topoisomerase that is also catalytically active on RNA in vitro and in cells. Uniquely, TOP3B is primarily localized to the cytoplasm, binds to open reading frames of mRNA, and regulates mRNA stability and translation in a transcript-specific manner. A common approach for studying TOP3B activity in cells is immunodetection of TOP3B•RNA covalent intermediates after bulk RNA isolation. However, in this approach, the RNA species is unknown and is not selective for the major TOP3B substrate, mRNA. In this protocol, we describe a recently developed and optimized protocol for capturing TOP3B•mRNA covalent intermediates using oligo-dT isolation of mRNA under protein-denaturing conditions. Covalent intermediates are then detected by a dual membrane slot blotting strategy with nitrocellulose and positively charged nylon membranes. Nitrocellulose membrane-bound TOP3B•mRNA covalent intermediates are analyzed by immunodetection, and nylon membrane-bound free mRNA is stained with methylene blue. The protocol detailed below has been validated with wildtype and mutant 3xFLAG-tagged TOP3B expressed in Neuro2A cells, with additional optimization for slot blotting using recombinant EGFP. Key features • This protocol is optimized for isolation of TOP3B•mRNA covalent intermediates from cultured mammalian cells. • Slot blotting allows for higher throughput and sensitive detection of TOP3B•mRNA covalent intermediates and allows for free mRNA to serve as a loading control. • Alternative to laborious extraction methods that do not select for TOP3B covalently linked to mRNA over other RNA species. • Can be completed in two days (not including variable time for mammalian cell sample collection).