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, 430 (4), 524-536

Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II

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Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II

Clarisse van der Feltz et al. J Mol Biol.

Abstract

In yeast, the U2 small nuclear ribonucleic acid (snRNA) component of the spliceosome is targeted for additional post-transcriptional modifications in response to cellular stress. Uridines 56 and 93 are both modified to pseudouridines (Ψ) during nutrient deprivation, while U56 is also pseudouridylated during heat shock. Both positions are located within stem II, which must toggle between two mutually exclusive structures during splicing. Stem IIa forms during spliceosome assembly, and stem IIc forms during the catalytic steps. We have studied how uridine 56 and 93 pseudouridylation impacts conformational switching of stem II. Using single-molecule Förster resonance energy transfer, we show that Ψ56 dampens conformational dynamics of stem II and stabilizes stem IIc. In contrast, Ψ93 increases dynamics of non-stem IIc conformations. Pseudouridylation impacts conformational switching of stem II by Mg2+ or the U2 protein Cus2; however, when Mg2+ and Cus2 are used in combination, the impacts of pseudouridylation can be suppressed. These results show that stress-induced post-transcriptional modification of U56 and U93 alters snRNA conformational dynamics by distinct mechanisms and that protein and metal cofactors of the spliceosome alter how snRNAs respond to these modifications.

Keywords: dynamics; pseudouridine; single-molecule FRET; snRNA; spliceosome.

Figures

Figure 1
Figure 1
Schematic of U2 stem II model RNAs used in smFRET experiments. (a) Chemical structures of uridine, pseudouridine, ribothymidine. (b) U2 stem II fluctuates between IIa (left) and IIc (right) basepairing. The locations of fluorophore attachment (stars) and pseudouridine incorporation (Ψ) are shown. The green and red stars indicated the donor (Cy3) and acceptor (Cy5) FRET fluorophores, respectively. Basepairing schemes depict stem II conformations observed in the spliceosome by cryo-electron microscopy (cryo-EM) before activation (stem IIa) and after the first catalytic step (stem IIc) [39, 42].
Figure 2
Figure 2
Representative single molecule fluorescence data for U2 stem II model RNAs. (a, c, e, g) Fluorescence intensity data collected from a single RNA molecule by excitation of the Cy3 FRET donor and by simultaneously monitoring Cy3 (green) and Cy5 (red) fluorescence emission and reported in arbitrary fluorescence units (A.U.). Anti-correlated changes in Cy3 and Cy5 fluorescence intensity are observed for both the WT RNA (a) and each variant containing the indicated Ψ bases (c, e, g). (b, d, f, h) EFRET values calculated from raw fluorescence data shown in (a, c, e, g) for each RNA molecule.
Figure 3
Figure 3
Influence of pseudouridylation on Mg2+-dependent conformational switching. (a, c, e, g) Histograms of EFRET values obtained from the indicated number (N) of single RNA molecules in the absence (filled, colored bars) or presence of 10 mM Mg2+ (black lines). (b,d,f,h) Changes in EFRET due to Mg2+ addition for each of the indicated RNAs. Each plot represents the result of subtraction of the histogram obtained in the absence of Mg2+ from the histogram obtained in the presence of Mg2+.
Figure 4
Figure 4
Influence of pseudouridylation on Cus2-dependent conformational switching. (a, c, e, g) Histograms of EFRET values obtained from the indicated number (N) of single RNA molecules in the absence (filled, colored bars) or presence of 6 μM Cus2 (black lines). (b, d, f, h) Histograms of EFRET values obtained from the indicated number (N) of single RNA molecules in the absence (filled, colored bars) or presence of both 6 μM Cus2 and 10 mM Mg2+ (black lines).
Figure 5
Figure 5
The impact of pseudouridylation on stem II switching in comparison to the unmodified RNA. Each plot represents the result of subtraction of the histogram obtained from the WT RNA under the indicated condition from the histogram obtained using RNA containing one or more pseudouridines (see Fig. 3 and 4). Signals above zero indicate an increase in observances of those particular EFRET values due to pseudouridine incorporation, while signals below zero indicate a reduction in observances of those EFRET values.
Figure 6
Figure 6
Structural probing of U2 stem II RNAs by RNase T1 protection assay. (a) Denaturing polyacrylamide gel of 5′-[P32]-labeled stem II RNAs incubated with RNase T1. The lanes labeled “A”, “D”, and “N” are standards representing a RNA ladder generated by alkaline hydrolysis, cleavage carried out under RNA denaturing conditions to identify the position of each guanosine, and the native intact RNA, respectively. The position of each guanosine and stem II structure are noted beside the gel. (b) 2D RNA representation of stem IIa and IIc with guanosine nucleotides circled. RNase T1 preferentially targets single-stranded guanosines. An alternative stem IIc pairing conformation, the IIc extension, is shown in the inset. (c–h) Quantification of cleavage at each guanosine within the stem II RNA. Each bar represents the average from four replicates, while error bars represent ±S.D. Data were normalized to the total band intensity in each lane as well as to the extent of cleavage of the WT RNA in each replicate. The F-test was used to determine the variance between data sets, and the t-test was then used to calculate the p-values. For clarity, comparisons which did not display significant differences in cleavage are not indicated by brackets. p ≥ 0.05; *, 0.01<p<0.05; **, 0.001<p<0.01.

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