Identification of hammerhead ribozymes in all domains of life reveals novel structural variations

Abstract

Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of "type II" hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred.

Figure 1. Consensus secondary structure model for hammerhead ribozymes and the expanded phylogenetic distribution of this self-cleaving ribozyme class.

(A) Consensus sequence and secondary structure of the catalytic core of hammerhead ribozymes. Annotations are as described previously . (B) Distribution of hammerhead sequences among all domains of life. The chart entitled “old” (inset) represents all previously known non-identical hammerhead ribozyme sequences –, , , –. The “new” chart includes previously known examples as well as all additional non-identical hammerhead ribozymes found in this study. Chart sizes are scaled based on the number of unique sequences as indicated. The chart on the right reflects the distribution of a subset of hammerhead ribozymes (not to scale with charts to the left). Clades that for the first time have been found to carry hammerhead motifs are boxed in yellow. Note that a large number of the hammerheads that we consider new in this graphic have been recently published , , , but the sequences of many were not available at the time of writing.

Figure 2. Type II hammerhead and representative pseudoknot substructures in type I, II and III ribozymes from diverse sources.

(A) Consensus sequence and secondary structure of widespread type II hammerhead ribozymes identified in this study. A pseudoknot forms the tertiary contacts that are presumed to stabilize parallel orientation of stems I and II. (B) Sequences and secondary structures of four type II hammerhead ribozymes. Diagrams reflect the orientation of stems I and II in the catalytically active structure. Closed circles represent wobble base pairs and the open square and triangle represent a trans Hoogsteen/sugar-edge interaction . Arrowhead indicates cleavage site.

Figure 3. Examples of gene contexts of clustered hammerhead ribozymes.

Hammerhead types (I, II or III) are indicated. Transcription from left to right is predicted for individual genes and operons, except in cases where arrows denote the opposite gene orientation. Genes, including those that encode hypothetical proteins (hyp), are labeled according to their respective genome annotations.

Figure 4. Mutational analysis of a metagenome-derived bimolecular hammerhead construct containing a one-base-pair stem II.

The indicated k _obs values were established in ribozyme reaction buffer containing 0.5 mM Mg²⁺ with incubation at 23°C. Deletions are designated by a delta symbol. Other notations are as described in Figure 2.

Figure 5. Rare nucleotide variations observed in the cores of some hammerhead ribozymes.

(A) Consensus secondary structure of the hammerhead core with highly conserved residues in yellow and variable residues in gray. Blue letters designate active natural variants tested previously. Red and green letters designate natural variations tested in this study that are expected to have deleterious effects or neutral/compensatory effects, respectively, on ribozyme function. (B) Atomic-resolution structure of portions of the Schistosoma mansoni hammerhead core. Colors are as defined in (A), with the addition of yellow designating strictly conserved nucleotides (built from PDB accession 2GOZ with pymol [66]). Stem I is in cyan, stem II in red, and arrows indicate position of insertions. Dashed lines in red and green represent hydrogen bonds that are expected to be disrupted or maintained, respectively. Other notations are as described for Figure 2. For complete secondary structure and additional information on these variants see Figure S6 and Figure S7 for variants that were inactive.

Figure 6. Variant hammerhead ribozymes encoded in saltern-derived DNA.

(A) Secondary structure models of variants HHmeta and HHphage. Annotations are as described for Figures 2 and 5. Residues corresponding to the highly atypical insertions are numbered 2a and 2b. Guanosine residues depicted in lowercase were added to facilitate transcription in vitro. (B) Effect of MgCl₂ concentration on the k _obs of HHmeta. k _obs values were determined in the absence of KCl (open circles) or in the presence of 3 M KCl (filled circles).

Figure 7. Two conserved human hammerhead ribozymes.

(A) Hammerhead from human C10orf118 intron with nucleotide substitutions and insertions occurring in pig shown in green. Variations observed in other mammals are in gray. Guanosine residues depicted in lowercase were added to facilitate transcription in vitro. (B) Hammerhead from human RECK intron with nucleotide variations observed in other mammals and birds in gray. Sequence with pink background highlights identical nucleotides between C10 and RECK hammerhead sequences. Other notations are as in Figure 2. (C) Self-cleavage during transcription in vitro of RECK and C10orf118 hammerhead ribozyme sequences from human and pig. The pig and human RECK hammerhead ribozymes are identical. Expected nucleotide lengths of RNA precursors and 5′ cleavage products are shown. First and last five nucleotides of RECK in (B) are depicted to illustrate boundaries of conservation, but are not part of the transcript. (D) and (E) Genetic contexts of the human hammerheads. Untranslated region (UTR) is colored in gray and coding sequence (CDS) in blue. Gene organization is not to scale, size of hammerhead-containing introns is according to NCBI annotation (build 37).