New classes of self-cleaving ribozymes revealed by comparative genomics analysis

Abstract

Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evidenced by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein we exploited this association to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. We identified three new self-cleaving classes, which we named twister sister, pistol and hatchet, from this collection, suggesting that even more ribozymes remain hidden in modern cells.

Figure 1. Self-cleaving ribozyme candidates

(a) Consensus sequence and secondary structure model for distinct variants of hammerhead ribozymes. R and Y represent purine and pyrimidine nucleotides, respectively. The site of ribozyme-mediated RNA cleavage (Clv) is identified by arrowhead. (b) Consensus sequence and secondary structure model for distinct variants of newly found variants of HDV ribozymes. (c) Consensus models for twister and twister sister ribozymes. Noncanonical base pairs and other additional structural interactions for twister ribozymes recently revealed by biophysical studies are not included.

Figure 2. Activity of a bimolecular twister sister ribozyme

(a) Sequence and secondary structure model of a bimolecular construct derived from TS-1 (Supplementary Fig. 1). Highly conserved nucleotides are depicted in red and Clv designates the cleavage site. Lowercase letters identify non-native guanosine residues that were added to facilitate in vitro transcription. (b) PAGE separation of bimolecular TS-1 ribozyme assay products demonstrating the requirement for the enzyme strand and for divalent metal ions. S designates the 5′ ³²P-labeled 23-nucleotide RNA substrate and 5′ Clv identifies the cleavage product. Reactions were conducted as described (see Online Methods) with variations noted. (c) PAGE separation of ribozyme cleavage products. S designates the 5′ ³²P-labeled 23-nucleotide RNA substrate, and C13 identifies the 5′ ³²P-labeled fragment band produced by incubation with excess unlabeled ribozyme for 30 min (Rxn lane). NR, T1 and ⁻OH lanes designate no reaction, RNase T1 partial digestion (cleaves after G nucleotides), or partial alkaline digestion (cleaves all internucleotides), respectively. (d) Mass spectrum analysis of bimolecular TS-1 cleavage reaction products were examined by mass spectrometry (see Online Methods). The second largest peak near the 5′ Clv annotation is the spontaneously formed opened version (observed mass, 4304.576) of the initial 2′,3′-cyclic phosphate product. Intensity is abbreviated int. (e) The dependence of twister sister rate constants on pH. (f) The dependence of Mg²⁺ concentration on twister sister rate constants. A version of this figure containing full-length gel images is shown in Supplementary Figure 11.

Figure 3. Structure and activity of pistol self-cleaving ribozymes

(a) Consensus sequence and secondary structure model for pistol self-cleaving ribozymes based on 449 unique examples. Annotations are as described in Fig 1a. (b) A bimolecular pistol ribozyme construct based on a representative from the bacterium Aliistipes putredinis. Annotations are as described in the legend for Fig. 2a. (c) Pistol ribozyme activity and cleavage site mapping of the A. putredinis bimolecular construct wherein the substrate RNA (S) was 5′-labeled with ³²P. Other annotations are as described in the legend to Fig. 2c. Trace amount of substrate was incubated with excess WT or M10 enzyme strand either with (+) or without (−) 20 mM MgCl₂. RNA cleavage products were separated by denaturing 20% PAGE. A version of this figure containing full-length gel images is shown in Supplementary Figure 12.

Figure 4. Structure and activity of hatchet self-cleaving ribozymes

(a) Consensus sequence and secondary structure model for hatchet self-cleaving ribozymes based on 159 unique examples. (b) A bimolecular hatchet ribozyme construct based on a representative from a metagenomic DNA sample. (c) Hatchet ribozyme activity and cleavage site mapping of the bimolecular hatchet ribozyme construct. A version of this figure containing full-length gel images is shown in Supplementary Figure 13.