Poster abstracts

Poster number 75 submitted by Jeff Levengood

Towards Developing a Structure Based Mechanism of Splicing Repression by hnRNP A1 at ssA7 on HIV-1

Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University ), Carrie Rollins (Department of Chemistry, Case Western Reserve University ), Jennifer Meagher (Life Sciences Institute, University of Michigan), Jeanne Stuckey (Life Sciences Institute, University of Michigan), Blanton Tolbert (Department of Chemistry, Case Western Reserve University )

Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1, are used to regulate splicing at the various donor and acceptor sites along the viral genome. One such site regulated by hnRNP A1 is the conserved 3’ acceptor splice site A7 (ssA7). Silencing of splicing at this site is necessary in order to retain the Rev Responsive Element (RRE) in the adjacent tat/rev intron. The RRE is responsible for nuclear export of unspliced and partially spliced transcripts. .
Our research seeks to clarify the binding determinant of hnRNP A1 on ssA7 by developing a structural model that will correlate ssA7 structure to its splicing function. For this model we are using isolated domains of both hnRNP A1 and ssA7. The protein UP1 is composed of the two RRM domains of hnRNP A1. For ssA7, SL3 of the three stem loop structure is examined as this contains a high affinity UAG binding site for hnRNP A1. We previously solved the 3D solution structure of SL3 by NMR and found the UAG is located in a terminal heptaloop.
Two separate pathways are being pursued for the development of the UP1:ESS3 co-structure: X-ray crystallography and HADDOCK modeling. For the X-ray crystallography, conditions for obtaining crystals of the co-structure are currently being optimized.
HADDOCK is “an information-driven flexible docking approach for the modeling of biomolecular complexes.” We are deriving the points of contact between UP1 and ESS3 with both NMR and mutagenesis experiments. With NMR, NOEs give exact points of contact between the two molecules while chemical shift changes show which residues might be involved actively or passively in the binding. Residues involved in binding can also be identified through mutagenesis experiments that alter residues on either UP1 or ESS3. Isothermal Titration Calorimetry (ITC) experiments are performed to determine the effect of the mutation on the binding of the two molecules.

References:
Levengood, J.D., Rollins, C., Mishler, C. H. J., Johnson, C.A., Miner, G., Rajan, P., Znosko, B.M., and Tolbert, B.S. (2012) Solution Structure of the HIV-1 Exon Splicing Silencer 3. J. Mol. Biol. 415:680-698.

Keywords: RRM, Splicing, Structure