Using a yeast-based screening system for identifying strategies to improve site directed RNA editing
2Raymond & Beverly Sackler School of Physics & Astronomy, Tel Aviv University, Israel
Adenosine deaminase acting on RNA (ADAR) enzyme can naturally change the genetic information at the RNA levels and potentially lead to alteration of the protein sequence. Advances made in understanding the ADAR mode of action enabled the development of two main strategies for artificially guiding RNA editing to selected sites (site-directed RNA editing (SDRE)): 1. Redirecting endogenous ADAR enzymes by developing editable structures using antisense RNA oligonucleotides (ASOs), and 2. Utilizing ASOs as guides to deliver the catalytic domain of engineered ADARs. Since the determinants that govern ADAR activity are largely unknown, in many cases these attempts resulted in low editing efficiency. To identify strategies that improve SDRE, we employed the yeast Saccharomyces cerevisiae as a screening system, an organism whose origins precede the emergence of ADARs but can exogenously express the human enzyme. For the first approach, we demonstrated that the sequence containing the dsRNA structure of the NEIL1 gene can serve as a novel element that better recruits endogenous ADARs to the target adenosine. In another line of research, since the delivery of a large protein can be challenging, we were interested in identifying the ADAR domains that are critical for its editing activity, and showed that ADAR carrying only its second RNA binding motifs is significantly more active. Overall, we demonstrate that our yeast-based screening platform is an efficient model for identifying new strategies to redirect endogenous or exogenous ADARs, and form the framework for better future designs in appropriate mammalian systems, and ultimately in human patients.