Poster abstracts

Poster number 77 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), Jennifer Meagher (Life Sciences Institute, University of Michigan), Jeanne Stuckey (Life Sciences Institute, University of Michigan), Blanton S. 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. The ssA7 RNA composes three stem loops, and a divide and conquer approach has been taken to determine the method by which hnRNP A1 binds and propagates along its RNA substrate. The first domain of ssA7 that is being studied is SL3, which contains the ESS3 binding motif for hnRNP A1. The 3D solution structure of SL3 was solved by NMR, and it showed the UAG motif of ESS3 to be located in a terminal heptaloop.
Building on the solution structure of SL3, experiments have been undertaken to solve the co-structure of hnRNP A1 with SL3. Most of this work has been done using UP1, a protein that consists of the two RRM motifs of hnRNP A1, but lacks the C-terminal glycine rich domain that is responsible for oligomerization of the full length protein. A crystal structure has been obtained of UP1 bound with the trinucleotide AGU, a sequence equivalent to a portion of the SL3 heptaloop. This structure shows the RNA is bound in a pocket formed by aromatic residues of RRM1 and residues from the linker between the two RRM domains. Elements of this binding are supported by 1D saturation transfer difference and NOESY NMR experiments. A variety of strategies are currently being employed to try and obtain crystals of full length SL3 bound by UP1.

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.

Rollins, C., Levengood, J.D., Rife, B.D., Salemi, M., and Tolbert, B.S. (2014) Thermodynamic and Phylogenetic Insights into hnRNP A1 Recognition of the HIV-1 Exon Splicing Silencer 3 Element. Biochemistry 53(13):2172-84.

Keywords: RRM, Crystal Structure, HIV