Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme

Abstract

Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg(2+), the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb(3+)-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg(2+)ions. Up to eight Mg(2+)ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg(2+)promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg(2+)binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg(2+)for the nested double pseudoknot structure.

Keywords: CPEB3; Mg2+; NMR; metal ion; pseudoknot; ribozyme.

FIGURE 1.

Secondary structures of the genomic HDV ribozyme (A), the chimpanzee CPEB3 ribozyme (B), and the small model constructs P1, P2, and P4 (C) used for resonance assignment. P1 is shown in red, P1.1 in orange, P2 in green, P3 and L3 in dark blue, and P4 in light blue; the J1/2 and J4/2 linkers are shown in black, the catalytic cytosine is in boldface; the two nucleotides G25·U20 that form a wobble pair only in the presence of Mg²⁺ (for the CPEB3 ribozyme, see far infra) are circled in A and B; in B, the difference between the chimpanzee and human sequence is shown in gray (G30A substitution); (C) the nucleotides added to the natural sequences of P1, P2, and P4 are shown in gray.

FIGURE 2.

Cotranscriptional self-cleavage assay of a 5′-elongated human (A) and chimpanzee (B) CPEB3 ribozyme sequence. In vitro transcription/cleavage reactions were left to proceed for 1–7 h or 15–120 min for the human and chimpanzee sequence, respectively. The incubation time (A in hours, B in minutes) is specified on top of the lanes. Lane C contains the control sequence as in Figure 1B, transcribed from a nonelongated template. Lane D contains the two dyes bromophenol blue (BB) and xylene cyanol (XC). In addition, major bands in the transcription lanes are labeled with the putative lengths of constructs, i.e., uncleaved ribozyme (85mer), post-cleavage ribozyme (67mer), and the cleaved 18mer. Two further bands in the transcription lanes correspond to the double-stranded DNA template, the top strand TS (24mer) and the template strand OT (107mer).

FIGURE 3.

Imino proton region of the [¹H,¹H]-NOESY of the exchangeable protons of the chimpanzee CPEB3 ribozyme. The cross peaks are labeled in the color corresponding to the ribozyme domain. The spectrum was recorded at 278 K in H₂O (100 mM KCl, 10 µM EDTA, pH 6.8). Note that there is no indication for the G20·U25 wobble pair in the absence of Mg²⁺.

FIGURE 4.

J_NN HNN-COSY of the chimpanzee CPEB3 ribozyme imino groups. The peaks are labeled in the color corresponding to the ribozyme domain. The spectrum was recorded at 278 K in H₂O (100 mM KCl, 10 µM EDTA, pH 6.8).

FIGURE 5.

Metal ion-induced changes in the [¹H,¹H]-NOESY correlations of the chimpanzee CPEB3 ribozyme. (A) Superposition of the [¹H,¹H]-NOESY spectrum of nonchangeable protons in the absence of Mg²⁺ (yellow), in the presence of 5 mM Mg²⁺ (green), and 9 mM Mg²⁺ (blue). Cross peaks with highly pronounced shifts and cross peaks broadened below the detection limit are labeled. Resonances in F2 belong to H1′ and resonances in F1 belong to H6/8 unless otherwise indicated. The spectra were recorded at 298 K in D₂O (100 mM KCl, 10 µM EDTA, pD 6.8). (B) Mg²⁺ and [Co(NH₃)₆]³⁺ binding to the imino protons. Superposition of [¹H,¹H]-NOESY spectra recorded in the absence of metal ions (yellow), in the presence of 5 mM Mg²⁺ (green, shifted by –0.08 ppm in F2), and in the presence of 1.5 mM [Co(NH₃)₆]³⁺ (purple, shifted by –0.16 ppm in F2). Upon addition of Mg²⁺, new cross peaks appear (labeled in boldface). The upper part contains the spectral region, in which cross peaks between [Co(NH₃)₆]³⁺ and RNA protons are located. The spectra were recorded at 278 K in H₂O (100 mM KCl, 10 µM EDTA, pH 6.8).

FIGURE 6.

Mg²⁺-induced changes in the [¹H,¹⁵N]-TROSY (A) and the J_NN HNN-COSY (B) of the imino groups of the chimpanzee CPEB3 ribozyme. (A) The spectrum in the presence of 5 mM Mg²⁺ is shown in orange-green and is superimposed with the spectrum recorded in the absence of Mg²⁺ (blue, shifted by 1.5 ppm in F1 for better comparison). In A and B, peaks that appear after the addition of 5 mM Mg²⁺ are labeled in bold; resonances that disappear after the addition of Mg²⁺ are labeled in gray. All spectra were recorded at 278 K in H₂O (100 mM KCl, 10 µM EDTA, pH 6.8).

FIGURE 7.

Tb³⁺-induced hydrolytic cleavage sites in the human CPEB3 ribozyme indicate sites of Mg²⁺ binding. The ribozyme was incubated with different concentrations of TbCl₃ (indicated on top of the gel) for 30 min at 298 K. The resulting ribozyme fragments were separated by PAGE. The positions that correspond to the observed cleavage sites were determined with the help of the OH^– hydrolytic cleavage ladder (first lane) and the T1 enzymatic cleavage ladder (second lane). The nucleotides most strongly cleaved at 40 µM Tb³⁺ are indicated in bold. Regions of additional nucleotides being cleavage at this concentration are marked with a gray background.

FIGURE 8.

Location of metal ion binding sites in the HDV and human CPEB3 ribozymes. (A,B) Crystal structure of the inhibited HDV ribozyme, pdb entry 3NKB (Chen et al. 2010): Full structure (A) and a close up (B) of the active site. Mg²⁺ ions are shown as dark blue spheres, J4/2 is shown in red, and the G25·U20 wobble formed only in the presence of Mg²⁺ is shown in yellow. (C) Mg²⁺ binding sites in the CPEB3 ribozyme. Each color represents a different binding site. The difference between the chimpanzee and human sequence (G30A substitution) is shown in gray.