Mark R. Macbeth
223A Mellon Institute
Department of Biological Sciences
Carnegie Mellon University
4400 Fifth Avenue
Pittsburgh, PA 15213
Ph.D., University of Chicago
Postdoctoral Appointment, University of Utah
Research in my lab will focus on the biochemistry of adenosine deaminases that act on RNA (ADARs). These RNA editing enzymes are responsible for changing an adenosine residue (A) to an inosine residue (I) in the context of an RNA molecule that is largely double-stranded (Figure 1). Since inosine is read as guanosine (G) by ribosomes and other cellular machinery, the effect is an A to G point mutation. ADARs are essential for properly functioning neurons, and several substrates are pre- messenger RNAs that code for neuronal receptors and ligand gated ion channels, such as the serotonin receptor and glutamate receptor.
The adenosine residues targeted for editing are specifically selected, but little is understood about how an ADAR can select one adenosine from hundreds present in an RNA molecule. It is widely accepted that the structure of the substrate RNA molecule is a major factor contributing to editing site selection. A major goal of my laboratory is to further characterize the basis for substrate recognition biochemically, using mutagenesis, and structurally, using X-ray crystallography.
In addition to the biochemical characterization of the ADAR reaction, I will also study the regulation of ADAR activity. The activity of these enzymes appears to be tightly controlled, as rampant editing of mRNA can be deleterious. The crystal structure of the catalytic domain of an ADAR revealed the presence of a small cell signaling molecule, inositol hexakisphosphate (IP6, Figure 2). This interesting molecule is essential for RNA editing activity, as it is likely involved in allowing the ADAR protein to fold properly, and it has been proposed that IP6 acts as a switch that turns the enzyme on and off. Therefore, understanding the role of IP6 is a key component to understanding the regulation of RNA editing activity by an ADAR.
Figure 2. The crystal structure of the catalytic domain of human ADAR2,and the details of the IP6 and zinc ion interactions with active site residues.
Macbeth MR and Bass BL. Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies. In Methods in Enzymology, (ed. J.M. Gott) Elsevier Inc., 424:319-331, 2007.
Macbeth MR and Bass BL.The role of secondary structure defects in substrate recognition by an adenosine deaminase that acts on RNA. Manuscript in preparation.
Macbeth MR, Schubert HL, VanDemark AP, Lingam AT, Hill CP and Bass BL. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science, 309:1534-1539, 2005.
Macbeth MR, Lingam AT and Bass BL. Evidence for auto-inhibition by the N-terminus of hADAR2 and activation by dsRNA binding. RNA, 10: 1563-1571, 2004.
Haudenschild BL, Maydanovych O, Véliz EA, Macbeth MR, Bass BL and Beal PA. A transition state analog for an RNA-editing reaction. J. Am. Chem. Soc, 126:11213-11219, 2004.
Macbeth MR and Wool IG. The phenotype of mutations of G2655 in the Sarcin/Ricin domain of 23S Ribosomal RNA. J. Mol. Biol, 285:965-975, 1999.