Self-cleavage of human CPEB3 WT ribozyme. (A) Secondary structure of HDV ribozyme (40). (B) Secondary structure of CPEB3 ribozyme (14). (C) Equilibrium between Alt P1 and P1. See Fig. S3 for details. (D) Standard self-cleavage reactions initiated by addition of 10 mM MgCl₂. RNA was 5′-end labeled with [γ-³²P]GTP during transcription. Plots of f_cleaved versus time. Each data point is the average of at least two trials ± the standard error of the experiments. Data were well fit to double-exponential eq 2 (solid line, R² = 0.999). Poor fitting to single-exponential eq 1 is provided for comparison (dashed line, R² = 0.993). (E) Co-transcriptional self-cleavage reactions. RNA was body-labeled with [α-³²P]GTP during transcription. Plots of f_uncleaved versus time, where R/(R+P₁+P₂) represents the uncleaved fraction. Each data point is the average of at least two trials. Data for both −59/68 (●) and −8/68 (○) were fit separately to eq 3. In this fit, which is for −59/68, A in eq 3 was fixed at 0.08±0.007, as ascertained from plateau values (avg. ± s.d.) averaged across 2-8 h time points for both constructs. Inset shows that the rate of transcription was constant throughout the reaction, as demanded by eq 3. Gels for panels (D) and (E) are 10% denaturing.

Enzymatic structure mapping of human WT CPEB3 ribozyme. (A) Structure mapping of self-cleaved 1/68 WT ribozyme. Denaturing 12% polyacrylamide gel is shown. 5′- end-labeled RNA was treated with RNases as described in Materials and Methods. The first three lanes are denaturing conditions (DEN), and the others are native conditions (NATIVE). Labels are as follows; OH⁻, limited alkaline digest; T1, A, and V1 limited digests with RNases T1 (G-specific), A (pyrimidine-specific), and V1 (double strand-specific), respectively; C, control sample without enzyme. Sets of three decreasing concentrations of each ribonuclease are provided (see Materials and Methods for details), and those with single hit kinetics were used for structure assignments, which is the third lane in each set. Numbering refers to selected bands produced by limited digestion with RNase T1. For the self-cleaved ribozyme, positions of the pairing elements are delineated by vertical lines and labeled on the right-hand side. Note that RNase V1 cleavage products greater than ~10 nt migrate approximately 1 nt slower than other cleavages owing to absence of a 2′,3′-cyclic phosphate. Fragments shorter than 10 nt generated by RNase V1 cleavages have anomalous migrations relative to RNase T1 and hydrolysis lanes (41). There are additional bands at the very bottom of the gel (not shown) that did not align with the T1 ladder and likely represent 2′ and/or 3′ phosphate monoesters. (B) Structure mapping of precursor–8/68 WT ribozyme. The gel is labeled as described above for the cleaved ribozyme. Selected bands produced by limited digestion with RNase T1 are numbered. Lanes to the left of the numbering are for precursor RNA band R₁, and those to the right are for band R₂ (see Figure 2). Both of these bands had been purified from a native gel as described in Materials and Methods. (C) Single and double stranded cleavages for the self-cleaved form in panel A mapped to a secondary structure. This structure is largely consistent with structure mapping of the self-cleaved form of the ribozyme and with free energy minimizations using ILM (29, 30), as well as the structure proposed by Salehi-Ashtiani et al (14). Positions of cleavage by RNases T1, A, and V1 are shown using green circles, blue diamonds, and red triangles respectively. Symbol size is relative to cleavage intensity.

Self-cleavage of human CPEB3 WT ribozyme analyzed on a native gel. (A) Self-cleavage reaction of −8/68 fractionated on 10% native gel (Lanes 1 – 8). RNA was 5′-end labeled with [γ-³²P]GTP during transcription and reactions were initiated by addition of 10 mM MgCl₂. Full-length RNA partitions into precursor R₁ and R₂ bands, and upon self-cleavage, a −8/−1 product, P. Marker bands corresponding to native an non-native RNAs that were previously purified on a qualitative native gel, renatured at 50°C and fractionated on this native gel are shown in Lanes 9 and 10 respectively. (B) Time-dependent change in the three RNA species fractionated on native gels. Inset is a plot of f_cleaved versus time for the data in panel A, where P/(R₁+R₂+P) represents the cleaved fraction. Each data point is the average of at least two trials ± the standard error of the experiments. Data were fit to eq 1.

Self-cleavage of human and chimp CPEB3 WT and C–2A ribozymes. Standard (A) and co-transcriptional (B) self-cleavage reactions for human WT (●), human C–2A (○), chimp WT (▲), and chimp C–2A (△). Initiation of the reaction, generation of plots, and fitting of data were the same as in Figure 1. Human and chimp ribozyme constructs were −8/68.