Poster number 38 submitted by Aldrex Munsayac
Small-angle scattering techniques as a structural tool to study RNA:RNA complexes
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)
Non-coding RNAs (ncRNAs) play numerous roles to maintain cellular homeostasis, regulating gene expression at the levels of transcription, RNA processing, and translation.1 The functions of many ncRNAs depends on how their three-dimensional structure changes in response to changes in the cellular environment (concentration of metabolites and/or metal ions, temperature) and interactions with other biomolecules (proteins and other RNAs).2 In the foodborne pathogen Listeria monocytogenes, two regulatory RNA elements—the positive regulatory factor A (prfA) RNA thermosensor (RNAT) and S-Adenosyl methionine riboswitch element A (SreA)—work in tandem to regulate its virulence.3 However, the precise molecular mechanism governing this interaction remains unclear due to difficulties in elucidating RNA:RNA complex structures. Therefore, there is a need for new strategies to probe structural information of RNA:RNA complexes. Biomolecular small-angle X-ray/neutron scattering (SAXS/SANS) are powerful solution-based techniques that provides information on molecular masses, particle dimensions and interactions, and low-resolution conformations. SANS is particularly well-suited to study multicomponent systems; by systematically changing the deuteration level of either the macromolecule or buffer medium, specific parts within the molecule can be singled out.4 Using the well-characterized human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) kissing complex as a model complex, we report strides towards the development of SANS as a novel tool for structure determination of RNA-only complexes. We have acquired high-quality SAXS data of the HIV-1 DIS complex and are currently in the process of taking SANS measurements of the partially deuterated complex to establish the methodology, which will then be applied to the prfA RNAT:SreA complex to unveil the global rearrangements of the individual components. SANS, complemented with other biophysical and biochemical techniques, improves our understanding towards how RNAs can fold into higher order assemblies to carry out their functions.
1. Cech, T. R.; Steitz, J. A. The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones. Cell 2014, 157 (1), 77–94.
2. Ganser, L. R.; Kelly, M. L.; Herschlag, D.; Al-Hashimi, H. M. The Roles of Structural Dynamics in the Cellular Functions of RNAs. Nat. Rev. Mol. Cell Biol.
3. 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.
4. Hoogerheide, D. P.; Forsyth, V. T.; Brown, K. A. Neutron Scattering for Structural Biology. Phys. Today 2020, 73 (6), 36–42.
Keywords: Small-angle scattering, RNA-RNA complexes, Integrative structural biology