UC Davis

Adenosine deaminases acting on RNA (ADAR) are a family of enzymes responsible for the conversion of adenosine to inosine in double stranded RNA. Inosine selectively base pairs with cytidine and is recognized like guanosine by cellular machinery. A-to-I editing has a myriad of downstream effects and dysregulated editing by ADARs is observed in multiple neurological and oncogenic diseases. In humans there are three ADAR enzymes, named ADAR1, ADAR2 and ADAR3, each with their own unique polypeptide sequences and RNA substrate preferences. There is great interest in uncovering the structural basis of RNA editing by ADAR with the potential to accelerate the development of targeted therapeutics to restore homeostatic levels of RNA editing in diseased patients. This thesis describes the effort to understand the structural basis behind ADAR and RNA interactions. Chapter 1 will provide an overview of what is known about RNA editing and describe the ways in which it is implicated in human disease. Chapter 2 describes the structural elucidation of an asymmetric protein-protein dimer of ADAR and how its formation aids in forming an RNA-bound complex. The biophysical characteristics of this protein-protein dimer will also be discussed, in addition to the identification of key conserved amino acid residues that are important for the formation of the protein-protein dimer. Chapter 3 describes the work towards the structural elucidation of the deaminase domain of ADAR1 bound to double stranded RNA. Current strategies to solve the crystal structure of ADAR1 bound to RNA will be discussed. A low resolution data set was collected. Information gained from the low-resolution model, how it was generated and strategies to optimize crystallization and diffraction to obtain a high-quality diffraction dataset will also be discussed. Chapter 4 will describe structural studies of ADAR2’s deaminase domain and an RNA duplex with a non-ideal nearest neighbor. Additionally, this chapter will discuss collaborative efforts towards the study of full-length ADAR2 bound to a double stranded RNA will be discussed.