Poster abstracts
Poster number 107 submitted by Eimile Oakes
ADAR3 Inhibits RNA Editing at the Q/R site of GRIA2 Transcripts Though Direct Binding
Eimile Oakes (Genome, Cell and Developmental Biology Program, Indiana University), Ashley Anderson (Medical Sciences Program, Indiana University School of Medicine), Aaron A. Cohen-Gadol (Department of Neurosurgery, Indiana University, Goodman Campbell Brain and Spine), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)
Abstract:
RNA editing is a cellular process in which RNA-editing enzymes modify transcripts in a site-specific matter. Adenosine to Inosine RNA editing at the Q/R site of a transcript encoding the glutamate receptor subunit B (GRIA2) changes the amino acid encoded from glutamine to arginine; this modification alters the calcium permeability of the glutamate receptor, having profound effects on cellular physiology. 100% of GRIA2 transcripts edited in the human brain; and, loss of editing at this site results in death of the organism. A reduction of GRIA2 editing has been observed in glioblastoma patients and has been shown to contribute to increased tumorigenesis; however, GRIA2 editing is not always correlated with reduction of its primary editing enzyme, ADAR2. The goal of these studies is to determine how editing of GRIA2 is regulated and the mechanism of that regulation. Humans express an editing deficient ADAR family member, ADAR3; we hypothesized that ADAR3 may regulate RNA editing, focusing on editing of GRIA2 mRNA.
We have established that ADAR3 expression inhibits GRIA2 RNA editing in the glioblastoma cell line, U87, and an immortalized normal human astrocyte (NHA) cell line. Furthermore, knockdown of ADAR3 in NHA cells leads to significantly increased editing. To interrogate the biological mechanism of ADAR3 inhibition of RNA editing, I took a two-pronged approach, expressing ADAR3 with mutations in annotated domains to determine its mode of action and immunoprecipitating ADAR3 to elucidate protein and RNA interactions. Our findings indicate that the dsRNA binding domains of ADAR3 are required for inhibition of RNA editing and that ADAR3 binds directly to GRIA2 mRNA transcripts. As the GRIA2 transcript is specifically edited by ADAR2 at the Q/R site, we suggest that ADAR3 directly competes with ADAR2 for binding to GRIA2 transcript, inhibiting RNA editing. To investigate the clinical relevance of these findings, we measure ADAR2 and ADAR3 expression and GRIA2 editing in glioblastoma tumors and matched adjacent brain tissue. We found that GRIA2 editing is reduced and that ADAR3 is overexpressed in a majority of the tumor samples compared to the normal adjacent tissue, suggesting aberrant ADAR protein expression in glioblastoma tumors that is correlated with reduced GRIA2 editing.
Keywords: RNA editing, GRIA2, glioblastoma