Biochemical analysis of hatchet self-cleaving ribozymes

Abstract

Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg(2+) to promote RNA strand scission with a maximum rate constant of ∼4 min(-1). As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2'-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

Keywords: RNA cleavage; RNA processing; comparative sequence analysis; phosphoester transfer; phosphorothioate.

FIGURE 1.

Sequence, structure, and function of hatchet ribozymes. (A) Updated consensus sequence and secondary structure model for hatchet ribozymes based on 210 different representatives. The arrowhead defines the site of ribozyme-mediated cleavage. Image was prepared using the R2R program (Weinberg and Breaker 2011). (B) Sequence and secondary structure of the bimolecular RNA construct Ht-1 based on metagenomic sequence SRS017191_Baylor_scaffold_14517/1281–1081 except that the loop region of P4 was replaced by UUCG. Nucleotides in red correspond to the most highly conserved positions in the consensus sequence. Nucleotides forming the 5′ and 3′ flanks of the cleavage site are numbered −1 and 1, respectively. Note that two non-native base pairs were added to P1 (brackets) to facilitate detection of the 3′ cleavage fragment by mass spectrometry. (C) Confirmation of ribozyme activity of Ht-1 carrying the two additional base pairs in P1. 5′ ³²P-labeled substrate (∼5 nM) was incubated in the absence (−) or presence (+) of enzyme (∼100 nM) either in the absence or presence of 20 mM Mg²⁺ as indicated for each lane of the 20% PAGE gel. Additional details are described in Materials and Methods. The bands corresponding to the 16-nt substrate (sub) and the 8-nt 5′-cleavage product (5′-clv) are annotated accordingly. (D) Mass spectrometry analysis of a Ht-1 reaction depicting peaks corresponding closely with the calculated masses for 5′-clv and the corresponding 8-nt 3′-cleavage product annotated 3′-clv. The calculated (calc.) and observed (obs.) atomic masses for the cleavage products are presented. (E) Analysis of a Ht-1 construct with a 6-bp P1 and a 2′deoxyribonucleotide (dC) replacing the cytidine ribonucleotide (C) at position −1 of the substrate. Other annotations are as described in C.

FIGURE 2.

Structure and activity assays with hatchet construct Ht-2. (A) Sequence and secondary structure for the bimolecular construct Ht-2 derived from a metagenomic sequence. Mutations (M1, M3, and M5) and compensatory mutations (M2 and M4) that restore structure are boxed and occur at the locations indicated. Other annotations are as described in the legend to Figure 1B. (B) Ribozyme activities of WT or mutant Ht-2 constructs using a 5′ ³²P-labeled substrate RNA. (NR) The reaction with WT RNA at time 0 as prepared by adding stop buffer before the addition of Mg²⁺. The asterisk denotes a reaction mixture without added enzyme strand. Other annotations and reaction conditions are as described in the legend to Figure 1C. (C) An example of a time course assay for ribozyme cleavage used to establish a k_obs value under a single reaction condition. (D) Plot representing the dependence of k_obs on Mg²⁺ concentration. (E) Plot representing the dependence of k_obs on pH. Three different buffers were used to span the pH range from 5 to 9.

FIGURE 3.

The effect of diverse metal ions on hatchet ribozyme activity. (A) Single time-point assays evaluating the ability of various divalent metal ions to support Ht-2 ribozyme activity. Reactions were incubated with 1 mM of the divalent metal ion as indicated for 30 min. Other experimental details and annotations are as described in the legend to Figure 1C. (B) Ht-2 ribozyme activity with certain divalent metal ions at 0.5 mM in the absence or presence of Mg²⁺. Other details are as described in Figure 1A. (C) The effect of cobalt hexammine on the function of Ht-2. Reactions were incubated in the absence (−) or presence (+) of 5 mM [Co(NH₃)₆]³⁺ and/or 5 mM Mg²⁺ as indicated. Other experimental details and annotations are as described in the legend to Figure 1C. (D) Sites of sulfur substitution (shaded) to generate the Rp or Sp isoforms of the ribozyme substrate. (E) Mass spectrometry data including the calculated (calc.) mass of the phosphorothioate substrate and the observed (obs.) mass of the sample. (F) Plot of the natural logarithm of the fraction of substrate RNA remaining uncleaved versus incubation time. O and S designate unmodified and phosphorothioate substrate RNAs, respectively. Of note, 10 mM MgCl₂ or 10 mM MnCl₂ were present in the reaction mixtures. Other conditions were as described in the legend to Figure 1C.