ADAR2: Towards a Structural and Kinetic Understanding of RNA Editing

Abstract

Adenosine Deaminases that Act on RNA (ADARs) are a small family of enzymes found in metazoans which edit pre-mRNAs by modifying the base adenosine to inosine. This editing results in translational mutations as inosine is interpreted as guanosine by translational machinery. Two editing events occur in humans on the Ionotropic Glutamate Receptor (GluR) pre-mRNA and are known to modify its ion permeability and resensitization kinetics. ADARs promiscuously edit perfectly complementary dsRNAs, however, in the context of complex secondary and tertiary structure, ADARs gain significant specificity for their substrates. Toward understanding the elements of ADARs which drive specificity in the context of a wild type substrate, we sought to generate a co-crystal structure of human ADAR2 with RNA and determine the structure of a flexible loop of the catalytic domain to ascertain its function. Crystals of GluR-B RNA and an ADAR2 truncation PP-R2D have been produced in small scale screens, and work to produce larger crystals suitable for x-ray diffraction is ongoing. Several mutants of the ADAR2 catalytic domain (CD), S458G and R455A, have been solved. Both of these mutant structures give some insight into the previously undefined electron density of an unstructured loop located near the catalytic site. These structures remain incomplete, yet provide a better descriptor of the dynamicity of the loop. To understand the kinetic role of the loop we have replaced it with glycine and found that editing is strongly inhibited. Current work focuses on further mutations to the loop to determine its kinetic role. The results of these studies have shown that large complexes of ADAR2 and substrate mimics can be crystallized, which is fortuitous for further structure determination, and that the unstructured loop of the catalytic domain remains highly dynamic despite mutations to decrease mobility.