Poster abstracts

Poster number 2 submitted by Haider Al-Awadi

Determining the Role of ADR-1 as a Key Cofactor in RNA Editing.

Haider Al-Awadi (Medical Sciences Program, Indiana University School of Medicine), Suba Rajendren (Genome, Cell and Developmental Biology Program, Indiana University), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)

Abstract:
RNA editing is a normal cellular process in which cells make modifications to specific nucleotide sequences within an RNA molecule. The most prevalent form of RNA editing in animals is an Adenosine (A)-to-Inosine (I) base deamination. ADARs (adenosine deaminases acting on RNA) are a family of enzymes that catalyze the hydrolytic deamination of Adenosine-to-Inosine within double stranded RNA (dsRNA). A-to-I RNA editing is essential in mammals, whereas Caenorhabditis elegans lacking ADARs are viable. When RNA editing is faulty, tumors, schizophrenia, ALS, and other neurological disorders may result. My project focuses on characterizing the A-to-I editing machinery from C. elegans. Previous results from our laboratory indicated that ADR-2 is the editing enzyme in worms, but ADR-1 promotes editing by ADR-2 in vivo. ADR-1 is a double-stranded RNA binding protein that is related to the ADAR family, but lacks deamination ability. Using purified recombinant proteins, I am testing how ADR-1 affects RNA editing in vitro. Using a Thin Layer Chromatography (TLC) assay and radiolabeled ATP, editing levels can be observed and assessed in the presence and absence of recombinant ADR-1 and ADR-2 (produced in insect cells). The dsRNA substrate used is the lam-2 3’ UTR, a known C. elegans edited mRNA. TLC is a highly sensitive method for detecting Adenosine monophosphates (AMP) and Inosine monophosphates (IMP). I will determine the rate at which the deamination reaction occurs. To verify the interaction of the proteins is necessary for editing, various mutants will be used and editing levels are observed. The binding domains of ADR-1 and ADR-2 are mutated to determine the interaction of the proteins to the RNA and its effect on RNA editing. More insight behind the mechanism for RNA editing will allow us to regulate the deamination mechanism, which can lead to treatment.

Keywords: ADARs, Deamination, Celegans