Collections/Functional Nucleic Acids/Ribozymes

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Ribozymes: RNA catalysts mimic enzymes

RNA was discovered to possess catalytic activity in the early 1980s, when self-splicing introns (group I) and ribonuclease P were identified (Cech et al., 1981, Guerrier-Takada et al., 1983). These notions, coupled to insightful experiments, showed that peptidyl transferases are resistant to proteolytic treatments, giving evidence that RNA - as opposed to proteins - participates in the catalysis of peptide-bond formation (Noller et al., 1992). During that same decade, the first ribonucleic acid enzyme, dubbed ribozyme, was identified in the Tobacco ringspot virus - the “hammerhead ribozyme” (Prody et al., 1986). Similar discoveries followed, with the hepatitis D virus ribozyme (Sharmeen et al., 1988), Varkud satellite ribozyme (Saville & Collins, 1990), and gImS ribozyme (Barrick et al., 2004) being representative examples. More recently, bioinformatic and metagenomic approaches have enabled the identification of novel ribozymes (e.g. pistol, hatchet, and twister ribozymes [Z. Weinberg et al., 2015]), thereby furthering our understanding of their physical and chemical properties. Currently, ribozymes can be classified into three categories: self-cleaving, trans-cleaving, and splicing ribozymes (which include introns and are not discussed further).

Even though self-cleaving ribozymes differ in size and structure, their mechanism of action remains highly conserved (Breaker et al., 2003; C. E. Weinberg et al., 2019). In general (Fig. 4a), the catalytic centre carries out the cleavage following four classical principles: 1) the leaving domain undergoes an in-line arrangement in preparation for a nucleophilic attack, which is in turn carried out by a guanine-activated 2’ hydroxyl group (Seith et al., 2018); 2) the negative charge of the two non-bridging oxygens is neutralised; 3) the attacking 2’ -OH group is deprotonated, and 4) the negative charge of the 5’ oxygen atom is neutralised. On the other hand, trans-cleaving ribozymes require other RNA molecules as substrates for catalysis. In fact, most of these originate from self-cleaving ribozymes that are divided into two domains: the target, which contains the site of cleavage, and a second motif that hybridises the target domain (Carbonell et al., 2011; Saksmerprome et al., 2004) (Fig. 4b). Systems such as this have been used to cleave viroid RNAs (Carbonell et al., 2011), in a fashion that is reminiscent of CRISPR-Cas9 devices.

Synthetic biology has also benefited from the potential of ribozymes as highly versatile tools. Compared to protein-based enzymes, ribozymes have several favourable traits. For instance, they are highly modular (Fujita et al., 2009), offer ease of control and predictability in terms of structure, and can be rationally modified using sequence-based approaches (Dolan & Müller, 2014). As such, these catalytic RNA motifs are central components of other forms of functional nucleic acids such as riboswitches and aptazymes, in which nucleic acid cleavage takes place upon ligand binding. Usually, the activity of aptazymes is used to trigger the translation of protein reporters that have relevance in diagnostics and biosensing (Famulok et al., 2007; Mellin & Cossart, 2015).

The versatile and highly modular nature of ribozymes makes them attractive in genetic circuits. In 2016, team William and Mary used ribozymes as insulators in their genetic circuits. This was done by adding the RiboJ ribozyme in the 5’ untranslated region (UTR) of mRNA molecules (Fig. 4c). Through this they were able to overcome problems in gene expression related to the coding region of the genes. The incorporation of RiboJ just prior to the coding region was able to insulate the gene expression from upstream effects in the mRNA sequence, thereby tuning gene expression such that it is only dependent on the promoter used.

Figure 4: Ribozymes. a) Self-cleaving hammerhead ribozyme. The hammerhead ribozyme is capable of self-cleave in the presence of magnesium ions. A nucleophilic attack occurs between residues (shown in grey and green) which result in the dissociation of part of the RNA strand from the ribozyme. b) The principle of self-cleavage can be used to create programmable cleaving RNAs. The ribozyme strand contains domains that can target the molecule to a substrate strand, such as a viral RNA, and the same nucleophilic attack occurs to cleave the target RNA, thus inactivating it. c) The RiboJ system introduced by team William and Mary 2016. RiboJ is a self-cleaving ribozyme that self-cleaves upon transcription in order to insulate mRNA molecules from upstream effects in the mRNA sequence.

References

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