Discipline: Biological Sciences
Abigail Shilvock - Cornell University
Co-Author(s): Hachung Chung and Charles Rice, The Rockefeller University, New York, NY
Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the most prevalent type of double stranded RNA editing in mammals. ADAR is also known to negatively regulate interferon signaling and enhance viral replication Previous experiments with mice ADAR1-/- cells demonstrated ADAR1’s role in negative regulation of interferon (IFN) signaling, but the mechanism is not clear. To further investigate ADAR1’s role and mechanism of action in human cells, the CRISPR/Cas9 system was used to generate ADAR1-/- human embryonic kidney (HEK) 293T cell lines. We then transfected both ADAR1-/- and ADAR1+/+ cell lines with polyinosinic:polycytidylic acid (poly I:C)- a double strand RNA that triggers IFN signaling. We hypothesized that ADAR1-/- cell lines would exhibit dysregulated IFN signaling upon poly I:C transfection. Our experiments show that ADAR1+/+ and ADAR1-/- cell lines have comparable levels of mRNAs for interferon beta (IFNb) and retinoic acid-inducible gene 1 (Rig-I) – an IFN stimulated gene – upon poly I:C transfection. Future directions include testing IFN signaling upon other stimuli such as virus infection.
Furthermore, this result may indicate that ADAR1 plays a role in regulating IFN signaling in a cell type specific manner, emphasizing the need to investigate other cell types besides HEK 293T cells. In addition, trying to generate knockout cell lines of the two ADAR1 isoforms, ADAR1p150 and ADAR1p110, was an ongoing aspect of this project. Access to these knockouts will allow for further experimentation to distinguish the individual functions of these isoforms.
Funder Acknowledgement(s): NIH Research Project Grant Program (R01).
Faculty Advisor: Charles Rice,