Poster abstracts
Poster number 54 submitted by Julia Warrick
Evaluating covalent protein•mRNA complexes produced by the neurological disease-linked RNA topoisomerase TOP3B
Julia E. Warrick (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA ), Durga Attili (Department of Neurology, Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI 48109, USA), Trevor van Eeuwen (Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 10065 USA), Sami J. Barmada (Department of Neurology, Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI 48109, USA), Michael G. Kearse (Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA )
Abstract:
The loss or mutation of Topoisomerase 3β (TOP3B), the only known topoisomerase in humans with the ability to catalyze RNA strand passage reactions, is linked to autism spectrum disorder, intellectual disability, and schizophrenia. Consistent with its reported role in mRNA translation, TOP3B primarily localizes to the cytoplasm, associates with the active translation machinery and polysome-bound transcripts, and crosslinks primarily to the open reading frame of mRNAs. Previous ribosome profiling experiments using HCT116 cells harboring various alleles suggest that TOP3B regulates mRNA stability and translation in both a topoisomerase-dependent and -independent manner. However, the basic molecular specificity of TOP3B topoisomerase activity and how de novo neurological disease mutations affect TOP3B activity and translational control are fundamental gaps in the field. Here, we report new data describing how one de novo mutation affects the topoisomerase activity of TOP3B on mRNA in Neuro2A cells. Using a newly developed TOP3B•mRNA activity assay, we determined that the autism-linked C666R mutant phenocopies a synthetic self-trapping R338W mutant, suggesting that the C666R mutation results in reduced ability of TOP3B to resolve covalent intermediates. In assessing the C666 residue within the predicted TOP3B structure, we provide evidence that the C666R mutation causes accumulation of unresolved TOP3B•mRNA covalent intermediates by directly disrupting metal coordination via a unique D1C3-type zinc binding motif within the zinc finger domain. Additionally, published literature has shown that prolonged overexpression of the synthetic self-trapping TOP3B-R338W mutant leads to cell death. We show that primary neurons are sensitive to high levels of unresolved TOP3B•mRNA covalent intermediates produced by the synthetic R338W mutant and that such adducts lead to ribosome collisions. In total, this work has begun to shed light on a new facet of RNA biology controlled by the RNA topoisomerase TOP3B and provides critical insights into neurological disease mutations.
Keywords: RNA-binding protein, neurological disease , RNA topoisomerase