Poster abstracts

Poster number 42 submitted by Jeff Levengood

RRM recognition of HIV-1 splice site A7

Jeffrey Levengood (Department of Chemistry and Biochemistry, Miami University), Carrie Rollins (Department of Chemistry and Biochemistry, Miami University), Clay Mishler (Department of Chemistry and Biochemistry, Miami University), Nitika Dewan (Department of Chemistry and Biochemistry, Miami University), Brent Kochert (Department of Chemistry and Biochemistry, Miami University), Blanton Tolbert (Department of Chemistry and Biochemistry, Miami University)

Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1 and ASF/SF2, are used to regulate splicing at the various donor and acceptor sites along the genome. ASF/SF2 is a splicing activator while hnRNP A1 is a splicing silencer. One such site regulated by these cellular proteins 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.
The ssA7 RNA structure contains three stem loops (SL1, SL2, and SL3). Binding sites for hnRNP A1 are located on all three stem loops while SL2 is the only region of the splice site which contains a binding site for ASF/SF2. Both proteins bind the RNA through their RNA recognition motif (RRM) domains. The RRM domains are a family of proteins which contain similar structures and conserved sequences for RNA recognition. Our research focuses on the manner in which these two proteins, with identical domains for RNA binding, are able to recognize different sequences and structures of RNA. Our research has focused primarily on hnRNP A1 as most of the binding sites at ssA7 are for this protein.
The binding determinants of hnRNP A1 for the RNA have been examined for each side. NMR experiments have identified residues on the RRM domains of hnRNP A1 that are involved in binding. Solving of the 3D structure of SL3 has revealed the orientation of the UAG bases that hnRNP A1 recognizes while binding studies have been done with mutations of these UAG residues. These studies confirm the importance of the hnRNP A1 binding sequence and structural features involved in molecular recognition.

References:
Saliou, J., Bourgeois, C., Avadi-Ben Mena, L., Ropers D, Jacquenet S, Marchand V, Stevenin J, Branlant C. Role of RNA structure and protein factors in the control of HIV-1 splicing. Front. Biosci. (2009) 14:2714-2729.
Mayeda, A,, Munroe, S., Caceres, J., and Krainer, A. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. (1994) 13:5483-5495.
Marchand V, Méreau A, Jacquenet S, Thomas D, Mougin A, Gattoni R, Stévenin J, Branlant C. A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J. Mol. Biol. (2002) 323:629-652.
Ding, J., Hayashi, M.K., Zhang, Y. Crystal Structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. Gene Dev. (1999) 13:1102-1115.

Keywords: RRM, HIV , Splicing