Poster abstracts
Poster number 51 submitted by Siara Sandwith
The effect of DHX36/G4R1 on the transcription of an abnormal DNA sequence linked to ALS
Siara N. Sandwith (Department of Biology, Ball State University), Yi-Ju Tseng, A. Krans, K. Green, Stephen Goutman, Eva Feldman, Peter Todd (Department of Neurology, University of Michigan), Eric Routh, Y.H. Wang, James Vaughn (Department of Cancer Biology, Wake Forest School of Medicine), H. Raimer, J. Wang (Department of Biochemistry and Molecular Biology, John Hopkins School of Medicine), Adam Richardson, Antonio Chambers, Melissa Smaldino, Philip Smaldino (Department of Biology, Ball State University), Peter Beerbower (Department of Plant Sciences, North Dakota State University)
Abstract:
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results in the breakdown of motor neurons, leading to severe deficiencies in eating, breathing, and moving; 80% of all ALS patients succumb to the disease within 5 years of diagnosis. The most common ALS-linked mutation occurs in the C9ORF72 gene (C9), consisting of a GGGGCC-sequence which becomes repeated hundreds to thousands of times, compared to healthy individuals who have <30 repeats. The mutated C9 sequence folds into extensive G-quadruplex (G4) DNA structures, which form within guanine-rich DNA and RNA sequences. G4s generally provide negative regulation on transcription and translation. G4-helicases such as DHX36/G4R1 (aliases: RHAU), potentially provide positive regulation on these processes via unwinding G4s. C9-repeat G4-DNA structures are partially unwound and transcribed, and the resulting RNAs form toxic RNA foci that sequester RNA binding proteins. It is unknown if G4R1 binds to the C9-repeat expansion or if it affects C9-repeat transcription. We hypothesized that G4R1 directly binds to and unwinds C9-G4 DNA and increases the transcription of toxic C9-repeat RNAs. To test this, we expressed and isolated recombinant DHX36/G4R1 and perform gel mobility shift assays with C9-G4 DNA. Furthermore, we used a C9-repeat in vitro transcription assay to determine if DHX36/G4R1 affects C9-repeat transcription. We found that DHX36/G4R1 directly and preferentially binds to C9 G4 DNA and facilitates the transcription of toxic C9 RNA in vitro. These data suggest that DHX36/G4R1 has the biochemical capabilities to facilitate the production of toxic C9 repeat RNAs, which are observed in C9 patient cells.
Keywords: G-quadruplexes, DHX36G4R1, ALS