Investigating the Effects of FUS and EWSR1 Proteins on T7 and RNA POL II Transcription

Abstract

Fused in Sarcoma (FUS), Ewing Sarcoma (EWSR1), and TATA-Box Binding Protein Associated Factor 15 (TAF15) make up the ubiquitously expressed FET family of proteins, which plays a large role in regulating genomic integrity, nucleic acid metabolism, and transcription. In FUS, mutations are known to result in neurodegenerative disorders such as frontotemporal dementia and amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. With EWSR1, a chromosomal translocation event with the transcription factor FLI1 results in the fusion protein EWS-FLI1, which is responsible for the titular Ewing sarcoma, an aggressive pediatric bone cancer. Placing FUS in a novel, in-vitro environment with the bacteriophage RNA polymerase T7 revealed an unexplored mechanism of RNA upregulation through the prevention of DNA:RNA hybrids (R-loops), a triple-stranded nucleic acid structure that forms during transcription. In Ewing Sarcoma and control cell lines, knockdowns and transfections have also identified how EWS-FLI1 drive tumorigenesis through the upregulation and downregulation of thousands of genes as identified through RNA sequencing. Here we combined a computational and biochemical approach to study how EWS-FLI1 influences transcription to promote oncogenesis, and how FUS promotes transcription through via DNA:RNA hybrids at the molecular level.