Structural Analysis of Multi-Helical RNAs by NMR–SAXS/WAXS: Application to the U4/U6 di-snRNA. Issue 5 (27th February 2016)

• Record Type: Journal Article
• Authors: Cornilescu, Gabriel; Didychuk, Allison L.; Rodgers, Margaret L.; Michael, Lauren A.; Burke, Jordan E.; Montemayor, Eric J.; Hoskins, Aaron A.; Butcher, Samuel E.
• Is Part Of: Journal of molecular biology. Volume 428:Issue 5 Part A(2016:Mar. 01)
• Page Start: 777
• Page End: 789
• Publication Date: 2016-02-27
• Subjects: smFRET single-molecule Förster resonance energy transfer -- FRET Förster resonance energy transfer -- cryo-EM cryo-electron microscopy -- MSA magnetic susceptibility anisotropy -- EDTA ethylenediaminetetraacetic acid; RNA -- NMR -- SAXS -- structure modeling -- spliceosome; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.11.026
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: NMR and SAXS ( s mall- a ngle X -ray s cattering)/WAXS ( w ide- a ngle X -ray s cattering) are highly complementary approaches for the analysis of RNA structure in solution. Here we describe an efficient NMR–SAXS/WAXS approach for structural investigation of multi-helical RNAs. We illustrate this approach by determining the overall fold of a 92-nt 3-helix junction from the U4/U6 di-snRNA. The U4/U6 di-snRNA is conserved in eukaryotes and is part of the U4/U6.U5 tri-snRNP, a large ribonucleoprotein complex that comprises a major subunit of the assembled spliceosome. Helical orientations can be determined by X-ray scattering data alone, but the addition of NMR RDC ( r esidual d ipolar c oupling) restraints improves the structure models. RDCs were measured in two different external alignment media and also by magnetic susceptibility anisotropy. The resulting alignment tensors are collinear, which is a previously noted problem for nucleic acids. Including WAXS data in the calculations produces models with significantly better fits to the scattering data. In solution, the U4/U6 di-snRNA forms a 3-helix junction with a planar Y-shaped structure and has no detectable tertiary interactions. Single-molecule Förster resonance energy transfer data support the observed topology. A comparison with the recently determined cryo-electron microscopy structure of the U4/U6.U5 tri-snRNP illustrates how proteins scaffold the RNA and dramatically alter the geometry of the U4/U6 3-helix junction. Graphical abstract: Highlights: A method is described for modeling RNA structures from NMR–SAXS/WAXS data. Inclusion of WAXS data significantly improves agreement with experimental data. Application to a 92-nt U4/U6 3-helix junction RNA is illustrated.