Novel ribozymes: discovery, catalytic mechanisms, and the quest to understand biological function

Abstract

Small endonucleolytic ribozymes promote the self-cleavage of their own phosphodiester backbone at a specific linkage. The structures of and the reactions catalysed by members of individual families have been studied in great detail in the past decades. In recent years, bioinformatics studies have uncovered a considerable number of new examples of known catalytic RNA motifs. Importantly, entirely novel ribozyme classes were also discovered, for most of which both structural and biochemical information became rapidly available. However, for the majority of the new ribozymes, which are found in the genomes of a variety of species, a biological function remains elusive. Here, we concentrate on the different approaches to find catalytic RNA motifs in sequence databases. We summarize the emerging principles of RNA catalysis as observed for small endonucleolytic ribozymes. Finally, we address the biological functions of those ribozymes, where relevant information is available and common themes on their cellular activities are emerging. We conclude by speculating on the possibility that the identification and characterization of proteins that we hypothesize to be endogenously associated with catalytic RNA might help in answering the ever-present question of the biological function of the growing number of genomically encoded, small endonucleolytic ribozymes.

Figure 1.

Reactions naturally catalysed by RNA. Two sequential transesterification reactions catalysed by group I. (A) and group II (B) introns in cis. These result in joined exons and linear and lariat introns, respectively. RNA hydrolysis catalysed in trans by the M1 RNA subunit of bacterial RNase P. (C) results in a phosphate containing 5′ end of the mature tRNA as the 3′ cleavage product (3′ P) and a 3′ hydroxyl group at the 5′ cleavage product (5′ P). Small nucleolytic ribozymes undergo transesterification reactions in cis (D), in which a specific 2′-hydroxyl attacks the neighbouring 3′,5′-phosphodiester bond. This results in a 2′,3′-cyclic phosphate and a 5′ hydroxyl at the 5′ and 3′ cleavage products, respectively. (E) Peptide bond formation catalysed in the ribosomal peptidyl transferase centre. This figure was adapted from (163).

Figure 2.

The cleavage and ligation mechanism of small nucleolytic ribozymes. The 2′ OH attacks nucleophilically the neighbouring 3′,5′-phosphodiester bond. Upon passing through a trigonal bipyramidal transition state, the cleavage reaction yields a 5′ product with a 2′,3′-cyclic phosphate and a 3′ product with a 5′ OH. In the reverse ligation reaction, this 5′ hydroxyl group attacks the 2′,3′-cyclic phosphate, and, passing through the same transition state, the two RNAs are joined by a conventional 3′, 5′ phosphodiester.

Figure 3.

Example secondary structures of the small nucleolytic ribozyme motifs. (A) The hammerhead ribozyme Ara2 (37), a type-III motif, featuring an open helix III. The inset shows the shapes of circularly permuted type-I (left) and type-II (right) hammerhead ribozymes with open helices I and II, respectively. (B) The hairpin ribozyme of tobacco ringspot virus satellite RNA (164). (C) The genomic hepatitis δ virus ribozyme (165). (D) The Varkud satellite ribozyme (166). (E) The glmS ribozyme (167) with the binding position of its cofactor glucosamine 6-phosphate (GlcN6P). (F) A type-P1 twister ribozyme from rice. The inset shows type-P3 (left) and type-P5 (right) permuted forms (23). Examples of a twister sister (G), hatchet (H) and a pistol (I) ribozymes (27). For each motif the helices are named or numbered in black according to the most-established nomenclature for that ribozyme class. This figure was drawn with R2R (168) and Adobe Illustrator.

Figure 4.

Catalytic strategies of the small nucleolytic ribozymes. To accelerate self-cleavage, the ribozyme motifs employ, to varying degrees, in-line arrangement (α), neutralization of the non-bridging pro-R_P (O_R) and pro-S_P (O_S) oxygen atoms (β), deprotonation of the attacking 2′ OH (γ) and neutralization of the negative charge at the 5′ oxygen atom (δ) (52,53). Additionally, the 2′ OH can be acidified by hydrogen bond donation to it (γ') or by preventing inhibitory interactions (γ''). Frequently, the N1 atom of a guanine contributes to the γ strategy, and the exocyclic amine of (sometimes the same) guanine to the γ' principle, while the functional groups involved in the realization of the β, γ'' and δ principles are more variable. For details see (54).

Figure 5.

Self-cleaving ribozymes support rolling circle replication mechanisms. (A) Symmetric rolling circle mechanism in viroids of the Avsunviroidae family and plant virus satellite RNAs. Circular (+) strand RNA is transcribed by DNA-dependent RNA polymerase into oligomeric (−) strand RNAs. Dotted lines indicate cleavage sites that define a single unit within the oligomeric RNA. Units are separated by hammerhead ribozyme cleavage and circularized by host enzymes, such as tRNA ligase. The circular (−) strand RNA is used for a second round of amplification yielding the (+) strand genome. In some plant virus satellite RNAs a hairpin instead of a hammerhead ribozyme could catalyse the cleavage of (+) strand oligomeric transcript, as well as potentially the ligation of the (+) strand linear monomer into a circular RNA. (B) Asymmetric rolling circle mechanism in plant virus satellite RNAs. First, the (+) strand RNA is transcribed by the host RNA polymerase into a long oligomeric transcript, the (−) strand RNA. Then the (−) strand RNA serves as template for a second transcription resulting in an oligomeric (+) strand. Hammerhead ribozymes, which are encoded in the (+) strand, cleave the oligomeric transcript into linear monomers. These unit-length transcripts are circularized either by ribozyme-mediated or enzymatic ligation.

Figure 6.

Several retrotransposons contain self-cleaving ribozymes. (A–D) The R2 element in Bombyx mori. (A) Organization of an rDNA unit with an R2 element inserted into a specific site within the 28S rDNA is shown. The external transcribed spacers (ETS) and internal transcribed spacers (ITS1 and ITS2) found in the precursor rRNA are depicted as light grey boxes. (B) Transcription of the R2 element in B. mori yields an RNA consisting of the HDV-like ribozyme in its 5′ UTR, the open reading frame (ORF) for the R2 protein and a 3′ UTR. (C) An expanded view of the R2 ORF illustrates a (simplified) composition of protein domains, which include zinc-finger and Myb-like nucleic acid binding motifs, the reverse transcriptase (RT) domain and the endonuclease domain (EN). Dotted lines designate untranslated regions. (D) Translation of the open reading frame generates R2 protein. Translation initiation likely occurs through IRES-like structure of the 5′ UTR. R2 proteins can bind the 5′ and 3′ end of the R2 element RNA. (E) Schematic representation of the simplified composition of L1Tc retrotransposons from Trypanosoma cruzi. The element is flanked by target site duplications (TSD) of usually 12 bp and it encodes a protein with an apurinic/apyrimidinic endonuclease (AP EN) domain, a reverse transcriptase (RT) and RNase H domain and a DNA binding domain. The first 77 nt of L1Tc harbor an HDV ribozyme (HDV). (F) Schematic representation of the composition of Penelope-like elements (PLEs). PLEs occur as tandem or multi-copy repeats in which an open reading frame (ORF) is flanked by Penelope long terminal repeats (PLTRs). The ORF encodes a protein with an RT and EN domain. The PLTRs contain a hammerhead ribozyme (HHR) and have been shown to also contain an intron in some PLEs. (G) Schematic representation of small interspersed nuclear element-like retrotransposons in Schistosoma often found in repetitive sequences. They consist of a promoter followed by a hammerhead ribozyme (HHR). All promoters could initiate transcription. (H) Schematic representation of the composition of retrozymes. Retrozymes are flanked by target site duplications (TSDs) and LTRs, which contain hammerhead ribozymes (HHR). The central region does not contain an open reading frame and is flanked by primer binding site (PBS) and poly-purine tract (PPT) elements needed for priming of DNA synthesis from the RNA element.