A widespread self-cleaving ribozyme class is revealed by bioinformatics

Abstract

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2,700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

Figure 1. Consensus sequence and secondary-structure model for twister self-cleaving ribozymes

(a) Detailed consensus model based on 2690 twister ribozymes depicted in its type P1 configuration, wherein the RNA chain begins and ends at the base of the P1 stem. The arrowhead identifies the cleavage site. Gray, black and red nucleotides designate conservation of at least 75, 90, and 97%, respectively; positions in which nucleotide identity is less conserved are represented by circles. Green shading denotes predicted base pairs supported by natural covariation. Notations i and ii identify predicted pseudoknots. Numbers in parentheses are the variable lengths of linker sequences that sometimes form stem structures as indicated. R and Y denote purine and pyrimidine, respectively. (b, c) The RNA chains of naturally occurring type P3 and type P5 configurations enter and depart the motif at the optional P3 or P5 stems, respectively.

Figure 2. Common associations between various genetic elements and twister or hammerhead RNAs

Plot of the ten genetic elements most frequently associated with twister type P1 (left) or hammerhead type II (right) RNAs as ranked by the fraction of occurrences of the element located within 6 kilobases (kb) of these RNAs. Other permuted ribozyme forms were not analyzed (see Supplementary Methods). Conserved protein domains from the Protein Families (pfam) or Clusters of Orthologous Groups (COG) databases with a stated relationship to Mu bacteriophages are designated. Protein domains of unknown function are labeled with a question mark. Genetic elements associated with both ribozyme classes are assigned a color, whereas other elements are depicted in gray.

Figure 3. Sequence, structure and activity of a twister ribozyme from N. vitripennis

(a) Sequence and secondary structure of the bimolecular construct derived from a twister ribozyme from N. vitripennis. Red nucleotides correspond to the ten most highly conserved nucleotides of the consensus sequence; other annotations are as described in the legend to Fig. 1a. Non-native guanosine residues were added to the 5′ end of the enzyme strand to facilitate transcription in vitro. (b) Activity of the N. vitripennis bimolecular ribozyme construct. Trace amounts of 5′ ³²P-labeled 22-nt substrate RNA (S) were incubated for 0 or 15 min in the absence (−) or presence (+) of 20 mM Mg²⁺ and the corresponding unlabeled RNA enzyme domain as indicated. 5′ ³²P-labeled cleavage product (5′ Clv) was separated from the uncleaved substrate by denaturing 20% PAGE. (c) For mapping the cleavage site, ³²P-labeled substrate (S) was partially digested with RNase T1 (T1; cleaves after G nucleotides) or with alkali (⁻OH), or was reacted with unlabeled enzyme RNA, followed by product separation using denaturing 20% PAGE. The products of the ribozyme reaction were loaded on the gel without (−) or with (+) prior acid treatment, or samples of each were combined (mix) to directly compare product mobilities. The asterisk denotes a band with a mobility that would be expected for a side product of RNA synthesis that is shorter by one nucleotide than the full-length substrate; this is not unusual for synthetic RNA preparations. A version of this figure containing full-length gel images is shown in Supplementary Figure 12.

Figure 4. Kinetic characteristics of a twister ribozyme derived from an environmental DNA sequence

(a) Representative time course for cleavage of a bimolecular twister ribozyme derived from an environmental sequence (Supplementary Fig. 5a). Other details are as described in the legend for Fig. 3b. (b) Effects of Mg²⁺ concentrations and pH on twister ribozyme rate constants. (c) Twister ribozyme activity assays in the presence of various divalent metal ions. 5′ ³²P-labeled substrate was incubated with excess enzyme RNA for 1 min in the absence (−) or presence of 1 mM of the divalent metal ions indicated. A version of this figure containing full-length gel images is shown in Supplementary Figure 13.