Poster abstracts

Poster number 61 submitted by Carrie Rollins

A thermodynamic study of the HIV acceptor splice site A7’s third stem loop, ESS3

Carrie Rollins (Department of Chemistry, Case Western Reserve University), Jeffrey Levengood (Department of Chemistry, Case Western Reserve University), Blanton Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
Alternative splicing plays a critical role in the lifecycle of the human immunodeficiency virus, transforming one strand of pre-mRNA into over forty different mRNA transcripts. Various combinations of acceptor and donor splice sites function to accomplish such variability, with the aid of host proteins to influence the assembly of the spliceosome. One such host protein is the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), known to abrogate spliceosome assembly by binding to highly conserved acceptor splice sites A2, A3, and A7. Splice site A7 is composed of three stem loops, intron splicing silencer (SL^ISS), exon splicing enhancer 3 (SL^ESE3), and exon splicing silencer 3, (SL^ESS3), all known to interact with hnRNP A1 through a yet undetermined mechanism. As a way to further define the protein-RNA binding mechanism, a mutational study was conducted to evaluate the role of nucleotides that compose the SL^ESS3 hairpin. For this study, the UP1 domain of hnRNP A1 was used. One dimensional 1H NMR and differential scanning calorimetry was used to define the structural and thermodynamic stability of each mutant, while electrophoretic mobility shift assays and isothermal titration calorimetry provided additional insight into the binding mechanism.

Keywords: HIV, alternative splicing