Poster abstracts

Poster number 56 submitted by Julia Warrick

Interrogating the RNA topoisomerase activity of the mRNA-binding protein TOP3B

Julia E. Warrick (Department of Biological Chemistry and Pharmacology; The Ohio State University, Columbus, OH ), Sarah E. Hoffmann-Weitsman (Department of Biological Chemistry and Pharmacology; The Ohio State University, Columbus, OH ), Michael G. Kearse (Department of Biological Chemistry and Pharmacology; The Ohio State University, Columbus, OH )

Abstract:
Topoisomerase 3β (TOP3B) is the only known RNA topoisomerase in eukaryotes and its loss or mutation contributes to intellectual disability, schizophrenia, and autism spectrum disorders. Tudor domain containing protein 3 (TDRD3) often forms a dimer with TOP3B and increases its topoisomerase activity and processivity, but TOP3B is active alone in vitro. Ribosome profiling of mammalian cells harboring catalytic (wildtype) or catalytically-dead alleles has shown that TOP3B regulates mRNA translation by two distinct modes: I) by acting as a traditional RNA-binding protein where catalytic activity is dispensable and II) by acting as an RNA topoisomerase where catalytic activity is required. How TOP3B selects its catalytic targets and why some mammalian mRNAs require an RNA topoisomerase rather than a classical RNA helicase remains enigmatic; however, the importance of TOP3B and its role in RNA biology is solidified by human neurological disease mutations and by its requirement for multiple human positive sense RNA viruses. We have focused our initial efforts on defining the molecular consequences of three mutations found in patients with neurological disease (P378Q, R472Q, and C666R) using newly developed assays to measure RNA topoisomerase activity in vitro and in mammalian cells. Additionally, using a self-trapping mutant to stabilize a covalently linked TOP3B•mRNA intermediate and denaturing His-tag pulldowns, we will identify all TOP3B mRNA substrates and the exact sites of topoisomerase activity to define sequence or structural preferences of TOP3B catalytic activity. In total, this work will shed light on a new facet of RNA biology controlled by the RNA topoisomerase TOP3B and could lead to the development of new therapeutic targets for neurodevelopmental disease.

Keywords: RNA topoisomerase, neurological disease