RRM
2012 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Alternative splicing is an essential step in the HIV life cycle. It is primarily regulated by host alternative splicing factors that bind different splice sites within the viral RNA genome, which function to repress or enhance splicing activity. Splice site A7 (ssA7, ~175nts), which is phylogenetically conserved, is composed of 3 stem loops: SL1, SL2, and SL3. These stem loops contain the ISS (SL1), ESE3 (SL2), and ESS3 (SL3) splicing regulatory elements (SRE). The host proteins hnRNP A1 and ASF bind to the SRE of ssA7 to repress or enhance splicing activities, respectively. Knowledge of the structural characteristics of ssA7 will serve to better understand the mechanisms of interaction between ssA7 and hnRNP A1/ ASF. We seek to determine a high resolution NMR structure of ssA7. Due to the inherent size limitation of NMR, the spectral analysis of large RNA constructs is cumbersome. To circumvent this challenge, we have decided to segmentally incorporate an isotope labeled SL3 fragment to an unlabeled SL12 fragment. Conditions were first optimized using synthetic DNA splints to form a well-defined ligation competent complex (LCC), as analyzed by non-denaturing PAGE. We found that both the length and the amount of splint are critical variables. However, the ligation yields were still very low. We hypothesized the problem might be the inefficient 5’-priming of SL3 with GMP. To explore this hypothesis, we developed a facile 1D 31P-NMR method to detect the relative amount of 5’-GMP SL3/5’-GTP SL3. The 1D 31P NMR spectrum of 5’-GMP gives rise to a well-resolved downfield phosphorus signal, which allows us to track the efficiency of 5’-GMP priming. In sum, we found that the length and amount of DNA splint affects LCC yields and we present a facile 1D 31P-NMR method for detecting 5’-GMP incorporation. This approach should prove useful to others who wish to prepare segmental labeled RNA contructs.
Keywords: HIV, Splice Site A7, ESS3
