Talk abstracts

Talk on Saturday 08:45-09:00am submitted by Jeffrey Levengood

Elucidating the Functional Significance of hnRNP A1 Binding to a RNA Structural Element of Human Enterovirus IRES

Jeffrey Levengood (Chemistry. Case Western Reserve University ), Mei-Ling Li (Molecular Genetics, Microbiology, and Immunology. Robert Wood Johnson Medical School), Michelle Tolbert (Chemistry. Case Western Reserve University), Blanton Tolbert (Chemistry. Case Western Reserve University)

Abstract:
Human Enterovirus 71 (EV71), the causative agent of hand, foot, and mouth disease, has been shown to recruit the small ribosomal unit through a type I IRES. Recent experiments have shown hnRNP A1 (A1) trans activates the EV71 translation by binding to stem loops II and VI. In an effort to better understand how A1 stimulates EV71 translation, we sought to characterize protein-SLII interactions using a combination of biophysical and functional biochemical assays.
The high affinity binding site for A1 is UAGGGU, with UAG being the primary binding element. Examination of the predicted secondary structure of SLII showed that a UAG element is located within a 5 nt bulge of the stem loop, which we hypothesized to be the specific A1 binding site.
A construct of EV71 SLII was in vitro transcribed and analyzed by 2D NMR spectroscopy. The 2D ¹H-¹H NOE patterns clearly show that the isolated SLII construct folds as predicted. Isothermal titration calorimetry (ITC) experiments were done with a truncated version of A1 (UP1), which contains the two RRM domains connected by a flexible linker. While it was initially assumed SLII had just one binding site for UP1, the experiments showed binding at two sites. Binding at both sites was found to be extremely tight, with binding at the first sight taking place in the picomolar range, and binding at the second sight taking place in the nanomolar range.
Since UP1 primarily recognizes single stranded RNA, it was hypothesized that the hairpin loop contains the second binding site. Mutations were introduced in both the bulge and hairpin loop. ITC experiments confirmed the involvement of both elements in UP1 binding, while NMR experiments showed that the mutations did not affect the RNA secondary structure. Furthermore, the significance of the bulge loop to IRES activity was probed using an in vivo translation assay. Substitution of the bulge UAG sequence with a CCC sequence reduced IRES activity by more than 80% relative to wild type levels.

References:
Shih, S.R., Stollar, V., and Li, M.L. (2011) Host Factors in EV71 Replication. J. Virol. 85(19):9658-9666.
Lin, J.Y., Shih, S.R., Pan, M., Li, C., Lue, C.F., Stollar, V., and Li, M.L. (2009) hnRNP A1 interacts with the 5’ Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication. J. Virol. 83(12):6106-6114.

Keywords: IRES, hnRNP A1