July 2010 Articles

RNA catalysts

Twenty five years ago the surprising discovery that certain RNA sequences catalyze chemical reactions ignited the hunt for RNA enzymes (ribozymes) and spurred intense investigation into the chemical and structural basis of RNA catalysis [1-3]. Since then, only a few naturally occurring ribozymes have been discovered. These include the small self-cleaving Varkud satellite (VS), hepatitis delta virus (HDV), hairpin, and hammerhead ribozymes found in selfish genetic elements such as satellite plasmids, viroids, and viruses [4-10]. Ribozyme activity resolves rolling circle replication intermediates through autocatalytic cleavage of RNA multimers followed by autocatalytic ligation of individual monomeric units into closed single-stranded circles.

Incredible progress has been made in understanding how RNA enzymes fold into complex structures and enhance the rate of chemical reactions [1]. All naturally occurring ribozymes catalyze phosphodiester transfer or hydrolysis with exquisite substrate specificity. A series of high-resolution crystal structures reveal the diversity of molecular architectures that enable specific substrate recognition and catalysis [1,11-14]. Corresponding biochemical experiments describe the relative contributions of individual nucleotides and chemical groups that contribute to folding and catalysis [15-21]. Helical junctions, non-Watson-Crick base pairs, pseudoknots, and metal ion-binding sites provide the foundation for RNA active sites capable of metal-assisted or general acid-base catalysis.

The observation that most extant ribozymes are found within selfish genetic elements or can trace their lineage through self-splicing mobile elements led to the hypothesis that ribozymes are modern vestigial remnants of an evolutionary era predominated by RNA ‘life’ forms [22]. This hypothesis led researchers to apply in vitro evolution technology (systematic evolution of ligands through exponential enrichment, or SELEX) to identify new artificial RNA catalysts, including ribozymes capable of RNA polymerization, peptide bond formation, tRNA charging, and many other biological activities required for primitive ‘life’ [23-26]. In contrast, the search for naturally occurring ribozymes moved forward at a relatively slow pace, suggesting that ribozymes may be rare entities - curiosities of unusual biological systems that have been largely discarded during evolution.

Regulatory ribozymes

The discovery in 2004 of a metabolite-sensitive ribozyme with a demonstrable role in gene regulation hinted at an expanded biological role for RNA catalysts [27]. The glmS (glutamine-fructose-6-phosphate amidotransferase) ribozyme/riboswitch, first identified in Bacillus subtilis and related Gram-positive bacteria, is found in the 5′ leader of transcripts that encode an enzyme necessary for the biosynthesis of glucosamine-6-phosphate, a cell wall precursor. The ribozyme binds to glucosamine-6-phosphate in order to activate self-cleavage at a defined position. Cleavage destabilizes the message, initiating a negative feedback loop that effectively reduces the biosynthesis of new glucosamine-6-phosphate [28]. This finding reinvigorated the search for new RNA enzymes.

Two years later, Szostak and colleagues [29] devised a clever selection approach to identify self-cleaving ribozymes in the human genome. The authors generated a library of single-stranded, closed circular genomic DNA fragments approximately 150 nucleotides in length. This library was used as a template for rolling circle in vitro transcription to generate long RNA multimers. After incubating the multimers at physiological salt concentrations, the authors purified sequences that were capable of self-cleavage but migrated at dimer length thus retaining one copy of the intact ribozyme. The recovered RNAs were used to generate the next round of circular DNA template. After 12 rounds of selection and amplification, they cloned and sequenced three autocatalytic self-cleaving ribozymes present in the OR4K15 (olfactory receptor family 4 subfamily K member 15), IGF1R (insulin-like growth factor receptor 1 gene), and CPEB3 (cytoplasmic polyadenylation element binding protein 3) genes and a fourth in a LINE 1 (long interspersed repetitive element 1) retrotransposon. This result clearly demonstrates that ribozymes are not as rare as initially believed.

Further characterization of the CPEB3 ribozyme demonstrated that it folds into a structure similar to the HDV ribozyme and catalyzes the same chemical reaction at the same relative position [29]. There is extensive secondary structure conservation between the two, but only six nucleotides are identical in the primary sequence. The CPEB3 ribozyme is conserved in mammals but is not found in other vertebrates. It is present in the second intron of the CPEB3 gene. Cleavage is hypothesized to regulate gene expression through destruction of pre-mRNA or possibly through formation of a truncated form of CPEB3 that lacks the N-terminal domain.

The extensive structural homology between ribozymes within a functional class and the absence of strong primary sequence identity led to a new bioinformatic strategy to identify ribozymes in genome sequences. The development of pattern-matching tools powerful enough to combine conserved sequence identity with secondary structural features into a single descriptor opened the door to genome-wide searches for new variations of the known ribozymes [30,31]. In a pioneering study, Przybilski and colleagues [32] used this approach to search the EMBL (European Molecular Biology Laboratory) database for self-cleaving hammerhead ribozymes. They identified two previously undiscovered ribozymes in chromosome IV of the Arabadopsis thaliana genome. The two ribozymes share conserved flanking sequences, are transcribed in a variety of plant tissues, and are capable of self-cleavage in vitro. The authors hypothesized that the ribozymes are not the result of viroid integration but instead evolved independently to perform a biological function, which to date has not been characterized. A clear outcome from this study is that pattern-based computational searches can successfully identify functional ribozyme variants that were previously hidden within a sequenced eukaryotic genome.