Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA

Abstract

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Figure 1

The structure of pseudouridine is shown. Secondary structure representations of the synthetic RNA hairpins based on the 1920 stem–loop region of E.coli 23S rRNA, in which positions 1911, 1915 or 1917 are modified with pseudouridine (Ψ), are shown. In the native rRNA, position 1915 contains a methylated pseudouridine (m³Ψ) (25). The positions have been renumbered consecutively from G₁ to C₁₉ for NMR analysis.

Figure 2

Representative normalized UV melting curves for the unmodified and modified RNAs taken in 15 mM NaCl, 20 mM sodium cacodylate, 0.5 mM EDTA, pH 7.0 are shown. The curve for unmodified RNA is shown in (A–D) (open squares), and the curves for modified RNAs (closed circles) Ψ1, Ψ2, Ψ3 and all-modified are shown in (A), (B), (C) and (D), respectively. The melting curves were normalized at 95°C.

Figure 3

CD spectra of the unmodified and modified RNAs. The molar ellipticities are normalized to RNA concentrations (3.7, 3.8, 4.3, 4.5 and 5.3 × 10^–6 M in molecules of RNA for unmodified, Ψ1, Ψ2, Ψ3 and all-modified, respectively). Each spectra is an average of four scans. The CD spectrum of the unmodified variant (open squares) is shown in (A–D) with overlays (closed circles) of the Ψ1, Ψ2, Ψ3 and all-modified spectra in (A), (B), (C) and (D), respectively. The total difference spectrum (Ψ1 + Ψ2 + Ψ3 – 2 × unmodified – all-modified) is shown in (D) (closed triangles).

Figure 4

The 1D imino proton (uridine H3/guanine H1/pseudouridine N1H and N3H) NMR spectra at 3°C of unmodified, Ψ1, Ψ2, Ψ3 and all-modified RNA sequences [from top to bottom (A–E)] dissolved in 90% H₂O and 10% D₂O (0.8–1 mM). The buffer employed for all samples contained 30 mM NaCl, 10 mM sodium phosphate and 0.5 mM Na₂EDTA, at pH 6.5. Nucleotide assignments based on 1D-NOE difference spectroscopy (Supplementary Material) are indicated above each resonance (the residues are numbered consecutively from G₁ to C₁₉).

Scheme 1. Synthesis of the 5′-O-BzH–2′-O-ACE-pseudouridine phosphoramidite 4: (i) TIPDSCl₂, pyridine, 0°C to room temperature; (ii) a) tris(2-acetoxyethyl)orthoformate, pyridinium p-toluenesulfonate, 4-(tert-butyldimethylsilyloxy)-3-penten-2-one, dioxane, 55°C; b) TEMED-HF, CH₃CN; (iii) BzH-Cl, diisopropylamine, CH₂Cl₂, 0°C; (iv) methyl tetraisopropylphosphorodiamidite, tetrazole, CH₂Cl₂, room temperature.