Poster abstracts

Poster number 74 submitted by Aldrex Munsayac

Towards structural characterization of RNA-only complexes via small-angle scattering techniques

Aldrex Munsayac (Department of Chemistry, University of Michigan), Ian Hall (Department of Chemistry, University of Michigan), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan)

Abstract:
Noncoding RNAs constitute the majority of the transcribed human genome and play a multitude of roles in biological processes; having been shown to regulate gene expression at the levels of transcription, RNA processing, and translation.1 In the foodborne pathogen Listeria monocytogenes, two riboregulators—the prfA RNA thermosensor (RNAT) and the SreA riboswitch—work in tandem to regulate the expression of PrfA, the major regulator of Listeria virulence gene expression.2 However, the precise molecular mechanisms governing the interaction between the prfA RNAT and SreA riboswitch remains undefined. In order to understand how these two cis-regulatory RNA elements are able to function in trans, elucidation of their structure at both the global and local scale is necessary. RNA-only structures account for only 1% of the three-dimensional structures deposited in the protein data bank.3 Furthermore, we expect that both the prfA RNAT and the SreA riboswitch undergo significant remodeling upon interaction, necessitating the development and implementation of new approaches for determining the structures of RNA-RNA complexes. To address this, we propose to employ a hybrid structural biology approach using small-angle X-ray/neutron scattering (SAXS/SANS) techniques complemented with NMR spectroscopy to unambiguously probe RNA-RNA interactions. Here, we lay the groundwork for the development of contrast variation SANS (CV-SANS) for RNA-only complexes. Using the HIV-1 RNA dimerization initiation site (DIS) kissing complex as a model system, we have collected SAXS data and successfully reconstructed the molecular envelope of the complex, which will serve as the foundation for our initial CV-SANS studies. CV-SANS coupled with NMR spectroscopy has the potential to solve very large, dynamic RNA structures.

References:
1. Cech, T.R.; Steitz, J.A. The noncoding RNA revolution—trashing old rules to forge new ones. Cell. 2014, 35, 77-94.

2. Loh, E.; Dussurget, O.; Gripenland, J;, Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell. 2009, 139(4), 770-779.

3. Das, R. RNA structure: a renaissance begins?. Nature Methods. 2021, 18(5), 439-439.

Keywords: Small-angle scattering, RNA-RNA interactions, RNA structural biology