Poster abstracts
Poster number 94 submitted by Christopher Morgan
Structural and mechanistic insights into hnRNP A1 - RNA recognition
Christopher E. Morgan (Department of Chemistry, Case Western Reserve University, Cleveland, OH), Jennifer Meagher (Life Sciences Institute, University of Michigan, Ann Arbor, MI), Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University, Cleveland, OH), James Delproposto, Jeanne Stuckey (Life Sciences Institute, University of Michigan, Ann Arbor, MI), Carrie Rollins (Department of Chemistry, Case Western Reserve University, Cleveland, OH), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University, Cleveland, OH)
Abstract:
In the HIV-1 genome, alternative splicing is a highly regulated process which is controlled through the utilization of individual suboptimal splice sites, where balanced usage of these splice sites is required for complete expression of the viral genome. At splice site acceptor 7 (ssA7), located at the 3’ end of the viral genome, splicing is inhibited through the binding of exon splicing silencer 3 (ESS3) by the hnRNP A1 protein. Many biochemical models suggest that the silencing effects of hnRNP A1 binding occur due to cooperative spreading and unwinding of the RNA; however, recent studies show that this protein binds specific regulatory RNA structural elements present within the HIV genome. In this study, the specificity of UP1 (the two RRM binding domain of the hnRNP A1 protein) to ESS3 is further defined by a crystal structure of the protein in complex with a short RNA oligomer, 5’ – AGU – 3’, analogous to the 3’ portion of the ESS3 apical loop. The structure revealed that UP1 uses a “nucleobase pocket” to make site specific contacts with the AG dinucleotide bases. This nucleobase pocket is formed by the inter-RRM linker and a beta sheet of RRM1 where RRM2 does not contact the RNA. This binding event between UP1 and ESS3 is kinetically characterized through the utilization of BioLayer Interferometry (BLI) and molecular dynamics simulations are performed in an attempt to further elucidate the binding mechanism of hnRNP A1 to RNA. Our kinetic studies show ESS3 mutants adversely affect UP1 association rates with little to no change in the off rates. These results imply that the ESS3 structure drives favorable UP1 interactions. In summary, this study provides critical insights into the biologically important UP1-ESS3 complex.
Keywords: HIV, hnRNP A1, ESS3