Talk abstracts

Talk on Saturday 11:10-11:25am submitted by Jeffrey Levengood

Elucidation of the binding determinants for hnRNP A1 on HIV-1 splice site A7

Jeffrey Levengood (Department of Chemistry and Biochemistry, Miami University), Clay Mishler (Department of Chemistry and Biochemistry, Miami University), Brittany Felder (Department of Chemistry and Biochemistry, Miami University), Elissa Falconer (Department of Chemistry and Biochemistry, Miami University), Nitika Dewan (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, are used to regulate splicing at the various donor and acceptor sites along the 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. Previous studies done on the binding of hnRNP A1 to ssA7 have resulted in two disparate mechanisms.
Our research seeks to clarify the binding determinant of hnRNP A1 on ssA7 by developing a model that will correlate ssA7 and hnRNP A1 structure to their splicing function. The 3D structure of the RNA composing the splice site is being examined by NMR while ITC is being used to study the thermodynamic model of hnRNP A1/ssA7 binding. NMR is also being used to examine which residues of hnRNP A1 are involved in the binding with ssA7.
The ssA7 structure contains three stem loops (SL1, SL2, and SL3). NMR has been used to determine the 3D structure of the third stem loop. For the ITC experiments, the hnRNP A1 analog UP1 was used. UP1 is composed of the two RNA-recognition motifs of hnRNP A1 and is missing the C-terminus, which is responsible for hnRNP A1 self-association. Preliminary ITC experiments examining the binding of UP1 along ssA7 showed that UP1 was able to assemble cooperatively along ssA7 without any self-association. A further truncated form of hnRNP A1, containing just the first RNA-recognition motif, was used in the NMR studies examining binding with SL3 of ssA7. Specific chemical shift changes were observed in the spectrum for residues of RRM1 involved in the binding with RNA. The chemical shift changes map to one surface of RRM1, containing the four stranded beta-pleated sheet and other elements of secondary structure.

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: HIV, Splicing, NMR