Structural Analysis of Multi-Helical RNAs by NMR–SAXS/WAXS: Application to the U4/U6 di-snRNA

Abstract

NMR and SAXS/WAXS 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-nucleotide 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 restraints improves the structure models. RDCs were measured in 2 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 FRET data support the observed topology. A comparison with the recently determined cryo-EM 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.

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

“…We annealed the WT U6 FRET reporter (U6 25-112 ) as well as each of the U6 mutants to U4 1-64 and monitored complex formation by native gel electrophoresis (Supplementary Figure S2B). In agreement with our previous experiments using a similarly truncated U4 (25), the 3′ region of U4 is not essential for stable U4/U6 complex formation with either U6 25-112 , U6 MUT1 or U6 MUT2 .…”

“…While previous data from us and others suggest that the three-helix junction formed between the U4 and U6 snRNAs is remarkably rigid in the absence of protein (24,25), we wondered if the peripheral regions of U6, those not base-paired with U4, are dynamic. These peripheral regions extend both 5′ and 3′ from the U6 domain that is base-paired with U4 in the di-snRNA and were not included in previous smFRET or nuclear magnetic resonance small-angle X-ray scattering experiments (24,25).…”

“…Complex formation was confirmed by native gel electrophoresis (Supplementary Figure S2A, lane 4), and the U4/U6 duplex RNA was analyzed by smFRET by immobilizing the U4/U6 duplexes and washing away any unbound RNA. Unexpectedly, instead of the static smFRET signal observed with U6 alone (Figure 1C) or the U4/U6 three-helix junction (24,25), 90% of U4/U6 molecules showed reversible transitions between two states with E FRET values of ∼0.2 and ∼0.7 (Figure 1D). The small number of remaining molecules that did not transition either exhibited a constant high E FRET of 0.9 consistent with absence of U4 (∼5%) or showed no transitions from the 0.2 E FRET state (∼5%).…”

“…These peripheral regions extend both 5′ and 3′ from the U6 domain that is base-paired with U4 in the di-snRNA and were not included in previous smFRET or nuclear magnetic resonance small-angle X-ray scattering experiments (24,25). We therefore created a U6 smFRET-reporter molecule containing the majority of the S. cerevisiae (hereafter ‘yeast’) U6 snRNA sequence (nt 25–112; U6 25-112 ) and a 20-nt, 3′ extension that terminates with a biotin for surface immobilization (Figure 1A).…”

RNA and Proteins: Mutual Respect

“…In contrast to the structure of the human tri-snRNP, in a structure of yeast tri-snRNP, a single-stranded region of U4 snRNA occupies the RNA-binding tunnel of Brr2 73, 80, 81 (illustrated in Figure 3). Is Brr2 now poised to completely separate U4 snRNA from U6 snRNA?…”