Talk abstracts

Talk on Friday 02:30-02:45pm submitted by Andrew Kehr

Structural insight into the editing of pre-mRNAs by human ADAR2

Andrew Kehr (Biological Sciences, Carnegie Mellon University), Dr. Mark Macbeth (Biological Sciences, Carnegie Mellon University)

Abstract:
Adenosine deaminases that act on RNA (ADARs) are a small family of proteins that modify dsRNAs in the mammalian brain. These enzymes edit adenosine to inosine, interpreted as guanosine by translational machinery, in serotonin and glutamate receptor B (GluR-B) pre-mRNAs in humans. Two editing events in the GluR-B RNA are known to modify ion channel permeability and resensitization kinetics. The enzyme that produces these mutations in humans is ADAR2, however, little is known concerning the high degree of specificity with which ADARs select their substrates. To better understand the elements of substrate selection and structurally predict other substrates we sought to 1) determine a co-crystal structure of ADAR2 with RNA and 2) determine the structure of the unstructured loop of the catalytic domain by making mutants in order to understand its function. We have generated crystals of GluR-B mimics with an ADAR2 truncation known as PP-R₂D with the help of an Art Robbins Phoenix LHS; screening to produce larger crystals suitable for x-ray diffraction is ongoing. We have solved two mutant structures, S458G and R455A, of the catalytic domain of ADAR2 to define a stable structure in an area of previously undefined electron density and identify the biochemical function of this unstructured loop of the protein near the site of catalysis. These structures remain incomplete, yet provide a better descriptor of the dynamicity of the loop. To further understand the kinetic role of the loop we have replaced it with several glycines and are comparing its kinetic activity to the wild type enzyme via poison primer assays employing a fluorescent label. 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.

Keywords: Adenosine Deaminase, RNA editing, Glutamate receptor