Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex

Abstract

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2-U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

Figure 1.

Emission spectra of Tb(III) and Cy3. With a delay time of 100 μs, all signal from the Cy3–Tb(III) sample at 565 nm was attributable to energy transfer from Tb(III) to Cy3 because Tb(III) does not emit at this wavelength. The lifetimes of the contributing components were the lifetimes of the Tb(III) emission in the presence of acceptors. Multiple D–A (donor–acceptor) distances resulted in multiple lifetimes. When recording the emission of Cy3, we did not incorporate the delay time of 0.1 ms used for Tb(III) measurements.

Figure 2.

Sequences of RNA oligomers used in this work. (A) Proposed complex formation between the central domains of human U2 and U6 snRNA. Previously established helices I, II, III and U6 intra-molecular stem loop (ISL) are labeled. The invariant top strand 32-mer area is shaded. (B) The simplified construct and its mutations used in the FRET study. The sequence represents the simplified original construct (called WT), which includes an invariant 32-nt top strand and a 69-nt bottom strand. Fluorescent dyes are attached at the 3′ or 5′ end of the 32-nt strand. The four mutation areas are shaded. All mutations were created by variations of segments of the bottom strand. ΔLOOP eliminates the ACAGAGA loop by mutating the bottom sequence to result in a complementary stem. ΔUG mutates the U–G base pair to U–A. Δ4W eliminates the four-way junction by replacing the shaded area with three adenosines. ΔU74 deletes the shaded uridine from the U6 ISL sequence.

Figure 3.

Demonstration of base pairing between top and bottom strands by a non-denaturing gel electrophoresis. Lane 1: the invariant top 32 mer. Lane 2: Δ4W bottom strand (40 nt) alone; Lane 3: Δ4W bottom with top strand. Both the brightest bands in lanes 1 and 2 disappeared in lane 3, indicating pairing of the strands. Lanes 4 and 5: WT bottom (69 nt) alone and with top strand. Lanes 6 and 7: ΔLOOP bottom (68 nt) alone and with top strand. Lanes 8 and 9: ΔU74 bottom (68 nt) alone and with top strand. Lanes 10 and 11: ΔUG bottom (68 nt) alone and with top 32 strand. The retardation of paired strands indicated the formation of base-paired complexes.

Figure 4.

Measurement of RNA metal-ion-binding stoichiometry by Job plot. By way of example, the Job plot for determination of ion-binding stoichiometry of the ΔU74 mutant complex (from which U74 had been deleted from the ISL) at pH 7.2 is shown. Luminescence was recorded for samples with a range of RNA/Tb(III) ratios, with total [RNA + Tb(III)] = 2 µM (see Materials and methods section for details), and was plotted versus RNA/(RNA + ion). Gaussian curve was fit to the data points and the peak of the curve was at RNA/[RNA + Tb(III)] = 0.32. The binding site number (ion/RNA) was n = (1–0.32)/(0.32) ∼2.1. The stoichiometry for other constructs was calculated with the same method (19).

Figure 5.

Calculation of the angle between helix I and U6 ISL using the acceptor normalization method. (A) The metal-ion-binding site on arm Y cannot be determined without knowing the angle between arms X and Y. As shown, the distance between Cy3 and a site-bound Tb(III) ion (dashed line from Cy3 to star) will correspond to different sites on arm Y if the angle between arms X and Y is unknown. (B) ‘Acceptor normalization method’ normalization process. Normalized Cy5 fluorescence, which is the acceptor intensity, is calculated from normalized Cy3–Cy5 emission spectra minus normalized Cy3 scan.

Figure 6.

The exponential decay fitting process used in the study. The exponential decay data were fitted with mono, bi- or tri-exponential decay curves. An example of the best fit in each category (1, 2 or 3 lifetimes) was presented. (A) WT construct at pH 5.6 best fitted to one-lifetime decay. τ ≈ 0.09 ms. (B) Construct ΔU74 at pH 7.2 best fit to two-lifetime exponential decay. τ₁ ≈ 0.12 ms; τ₂ ≈ 0.27 ms. (C) Construct WT at pH 7.2 best fitted to three-lifetime exponential decay. τ₁ ≈ 0.06 ms; τ₂ ≈ 0.22 ms; τ₃ ≈ 0.63 ms. The residuals represented the differences between experimental data and fitting curves, which showed the goodness of fit. The choice of the number of lifetimes is based on the R² and χ² values. The improvement of the fit was shown by comparing other fits and the residue figures. Residuals refer to the difference between theoretical and experimental data.