Mechanisms of catalytic RNA molecules

Abstract

Ribozymes are folded catalytic RNA molecules that perform important biological functions. Since the discovery of the first RNA with catalytic activity in 1982, a large number of ribozymes have been reported. While most catalytic RNA molecules act alone, some RNA-based catalysts, such as RNase P, the ribosome, and the spliceosome, need protein components to perform their functions in the cell. In the last decades, the structure and mechanism of several ribozymes have been studied in detail. Aside from the ribosome, which catalyzes peptide bond formation during protein synthesis, the majority of known ribozymes carry out mostly phosphoryl transfer reactions, notably transesterification or hydrolysis reactions. In this review, we describe the main features of the mechanisms of various types of ribozymes that can function with or without the help of proteins to perform their biological functions.

Introduction

Ribozymes, or catalytic RNA molecules, were discovered independently by Altman (1) and Cech (2), who found that RNAs can act as catalysts for chemical reactions, a notion that had been anticipated (3, 4). This discovery propelled the hypothesis, known as the RNA World Hypothesis (5), which proposes that life began with RNA or RNA-like molecules that can store genetic Information and perform catalysis. Since then, many more roles for RNA molecules have been discovered and the central role of RNA In biology Is now well established. To date, several classes of natural ribozymes have been identified including small self-cleaving RNAs, splicing molecules, RNase P, and the ribosome. Not all catalytic RNA molecules act alone, some RNA-based catalysts such as RNase P, the ribosome, and the spliceosome have evolved from their putative single-RNA ancestors to incorporate protein components to perform their function in cells. Most natural ribozymes catalyze phosphoryl transfer reactions to cleave and/or ligate the RNA phosphodiester backbone. The exception is the ribosome, which catalyzes peptide bond synthesis.

Self-cleaving RNA ribozymes

Self-cleaving ribozymes play roles in activities as diverse as mRNA biogenesis, gene regulation, and circular RNA replication (6). Recently, bioinformatics studies (7) have identified new catalytic RNA motifs, although their biological function remains unknown. Nevertheless, these studies show the potential for an even more widely spread role of self-cleaving ribozymes in biology.

Although each self-cleaving ribozyme adopts a unique three dimensional structure and follows a distinct catalytic and kinetic mechanism, they all catalyze a specific and reversible self-endonucleolytic trans-esterification cleavage reaction. Nucleolytic ribozymes use general acid-base catalysis, frequently performed by a nucleobase. Figure 1A illustrates the active site of the hammerhead ribozyme, an example of cleavage by general acid-base. Aside from the Hepatitis Delta Virus (HDV) and twister sister ribozymes (8), in all self-cleaving ribozymes a guanine acts as the general base, while the general acid is more variable. Nucleobases, either adenine or cytosine, hydrated Mg²⁺ (pistol ribozyme (9)), or a coenzyme¹ (glmS ribozyme (10–12)) can all act as the general acid. Divalent metal ions play an important role in folding all RNA molecules, but they are generally not required for catalysis. Metal ions may act as a cofactor to speed up the reaction and/or activate a catalyst. Unique among self-cleaving RNAs, the glmS ribozyme, which was discovered in a search for novel classes of riboswitches, uses an exogenous small molecule, glucosamine 6-phosphate (GlcN6P), as a catalytic cofactor. The glmS ribozyme self-cleaves using GlcN6P as a coenzyme and in this manner links its own degradation to gene expression (13).

Figure 1. Examples of different catalytic strategies in ribozymes.

A) Diagram illustrating the active site of the Schistosoma mansoni hammerhead ribozyme (PDB 3ZP8 (56)). The hammerhead ribozyme (grey) uses general acid base catalysis to cleave its substrate (yellow) through the involvement of a nucleobase (57). The structure contains a 2’-O-methyl to prevent cleavage (labeled O2’ in the figure). B) Diagram illustating the active site of a post-cleavage a complex of Thermotoga maritima RNase P (grey) in complex with tRNA (yellow) and 5’ leader (blue) (PDB 3Q1R (21)). The two magnesium ions are shown as green spheres.RNase P, like many other ribozymes, employs a two metal ion mechanism to cleave its target RNA. C) Diagram illustrating the peptidyltransference center (PTC) of the Haloarcula marismortui ribosome (PDB 1VQN (58)). The ribosome active site is composed solely of RNA. In the diagram, the 23S ribosomal subunit is shown in grey, cytidine-cytidine-adenosine-phenylalanine-caproic acid-biotin in the P-site is shown in gold, and cytidine-cytidine-hydroxypuromycin in the A-site is shown in blue. The α-amino group in the latter is replaced by an hydroxyl to slow down the reaction. The ribosome illustrates a ribozyme not involved in a phosphoryl transfer reaction.

The reaction mechanisms of all self-cleaving ribozymes differ at the atomic level due to the presence of specific catalysts and cofactors around the active site. However, in all cases, site-specific cleavage is an SN2 transesterification reaction of a 3’ 5’phosphodiester bond where reaction of the phosphodiester unit with its adjacent ribose 2’-hydroxyl group results in two RNA fragments, one carrying a 2’,3’-cyclic phosphate terminus and the other a 5’-hydroxyl terminus. The reaction is typically accelerated by at least one million-fold taking advantage of four general catalytic strategies (14). Not surprisingly, if the products are held in place, self-cleaving ribozymes can catalyze the reverse reaction (ligation).

Ribonuclease P (RNase P)

RNase P and the ribosome are two naturally occurring ribozymes present in all three domains of life. RNase P is a multi-turnover ribozyme that recognizes its substrate in trans without extensive base-pairing between the substrate and the ribozyme (15). RNase P is thought to be an ancient endonuclease and a relic of the primordial RNA world. Its major function is to catalyze the cleavage of the 5’ leader sequence from precursor tRNAs (pre-tRNAs), but several other RNAs have been demonstrated to be RNase P substrates as well (15). In contrast to the cleavage mechanism of small ribozymes, RNase P generates products with 5’-monophosphates and 3’-hydroxyls, similar to the cleavage by Group I and Group II self-splicing introns (see below).

RNase P is a ribonucleoprotein complex. Although the RNA component is responsible for catalysis, the presence of the protein component(s) increases the catalytic efficiency. The number of protein components in the complex varies and increases with the complexity of the organism. In bacteria, in addition to the single catalytic RNA component, RNase P contains one small protein subunit that plays a role in substrate recognition and enhances catalysis. The organization of RNase P in higher organisms is more diverse, forming a large ribonucleoprotein complex where the protein components form the majority of the complex, although there is always a single RNA component responsible for catalysis (16). Archaeal RNase Ps contain four to five proteins in addition to the RNA subunit (17), whereas eukaryotic RNase Ps contain nine to ten proteins (18, 19). Some of the archaeal and eukaryotic proteins are clearly related, but the bacterial protein appears to have limited similarity to the archaeal or eukaryotic proteins. The presence of a protein-only RNase P in plants and other organisms (PRORP) (20) demonstrates that replacing RNase P with a protein-only enzyme is possible. The reason for the increased complexity of the RNase P ribonucleoprotein complex in eukarya and archaea, as well as the persistence of the RNA component as the catalytic moiety, is still unknown (16).

A crystal structure of bacterial RNase P in complex with a tRNA substrate revealed the spatial organization of the ribozyme and the detailed mechanism by which RNase P hydrolyzes pre-tRNA to produce the required 5’-phosphorylated tRNA (21). The proposed mechanism was later confirmed by biochemical experiments (22). More recently, cryo-electron microscopy (cryoEM) structures of archaeal, yeast, and human RNase Ps alone and in complex with a tRNA substrate (17–19), as well as the closely related mitochondrial RNA processing ribonuclease (RNase MRP) complex (23), show the role of proteins in the ribonucleoprotein complex. The structures also confirm the central role of the RNA moiety in catalysis and the common catalytic mechanism of RNA cleavage. Additionally, these structures show how the protein selects the pre-tRNA substrate for proper positioning in the RNA-based active site and the role of metal ions in catalysis.

Sequence analysis uncovered several highly conserved sequence regions in the RNA component, suggesting a highly conserved architecture of the catalytic center. The conservation also suggested that the chemical mechanism of pre-tRNA processing is evolutionarily conserved. The structures of bacterial, archaeal, and eukaryotic RNase P confirm the common structure of the catalytic active site and mechanism. All structures show that the active site includes two Mg²⁺ coordinated by adjacent non-bridging oxygens in phosphate groups of the backbone of the RNA subunit, as well as the scissile phosphate group at the cleavage site of the pre-tRNA (Figure 1B). One of the Mg²⁺ ions is also coordinated by a conserved nucleotide base in the RNA component, apparently the only nucleobase that participates directly in catalysis. Another Mg²⁺ ion activates a water molecule to perform an SN2 type nucleophilic attack (transesterification reaction) to hydrolyze the phosphodiester backbone of the pre-tRNA substrate. Two metal ion nucleic acid cleavage mechanisms are not uncommon in proteins and ribozymes (15).

Ribosome

The ribosome is a large and highly complex cellular machine formed by two subunits: 30S and 50S subunits in bacteria, and 40S and 60S subunits in eukaryotes. Whereas the anticodons/codons interactions occur in the small subunit, the large subunit contains the peptidyl-transferase center (PTC), which catalyzes the formation of peptides by joining amino acids together to form protein chains. Protein synthesis is a complex, multistep process where numerous initiation, elongation and release factors ensure that protein synthesis occurs progressively, orderly, and with high specificity (24) .

Insights obtained from numerous electron microscopy and X-ray crystallography structures (for example (25–27)) have transformed our understanding of how the ribosome catalyzes protein synthesis. The ribosome catalyzes two chemical reactions: aminolysis to form peptide bonds during protein synthesis (peptidyl transferase reaction) and peptidyl hydrolysis to release the complete protein from the peptidyl tRNA upon completion of translation. Both reactions occur on the same site of the ribosome, the PTC (28). Although the ribosome is composed of proteins and ribosomal RNAs, the PTC active site is composed entirely of RNA, thus RNA is sufficient to catalyze the formation of the peptide bond. Even though proteins are not needed for catalytic activity, they act to maintain the active site integrity on the exterior of the cleavage site. The PTC includes the P site (where peptidyl tRNA is accommodated) and the A site (where the aminoacyl tRNAs bind) (Figure 1C). Both the A site and the P site interact with the 3’ terminal CCA sequence of the tRNAs (29). The peptidyl tRNA in the P site is protected from nucleophilic attack if the A site is empty, but upon binding of an aminoacyl tRNA to the A site there is a conformational change that makes the peptidyl tRNA susceptible to nucleophilic attack (30).

During elongation, the ribosome PTC catalyzes aminolysis of the ester bond, where the α-amino group of A-site aminoacyl-tRNA nucleophilically attacks the P-site peptidyl-tRNA at the carbonyl carbon of the ester bond that links the peptide to the tRNA (31). The reaction involves formation of a tetrahedral intermediate, following loss of the proton from the nucleophilic nitrogen to form the amide. The products of the reaction are a peptide that is one residue longer, ester-linked to the tRNA that carried the amino acid into the reaction, and a free tRNA. The ribosome accelerates this reaction by ~10⁶- to 10⁷-fold (32). In the second reaction, which occurs at the termination phase of protein synthesis (at the end of translation elongation), the carboxy-terminus of the polypeptide chain attached to the P-site tRNA undergoes nucleophilic attack at the ester carbon by a water molecule, which leads to release of the newly synthesized polypeptide. In this reaction, binding of release factors to the A site induces conformational changes similar to those for peptidyl transfer during translation elongation (33). The mechanism of peptide release closely resembles the mechanism of peptide bond formation, differing only in the identity of the nucleophile (water versus amine) (28). The reaction is thermodynamically favored towards peptide bond formation due to the higher energy of the aminoacyl-tRNA bond over the peptide bond (30).

Splicing ribozymes

Pre-mRNAs are composed of coding regions, called exons, and non-coding, intragenic regions, called introns. Introns are removed from precursor RNA transcripts to form mature RNA by splicing together the exons. While spliceosomal introns are found in eukaryotes and utilize protein-RNA complexes (spliceosomes) for splicing, group I and group II introns can self-splice without the help of any protein. These ribozymes facilitate their own excision and the ligation of the two flanking exons.

Group I intron ribozymes are found in small RNAs in bacterial, phage, viral, and organellar genomes and often in nuclear rDNA genes of fungi, plants, and algae (34). Several crystal structures of Group I introns have been reported (35, 36). Structurally, they contain a core region consisting of three helical stacks stabilized by peripheral elements, as has been observed in other large RNA molecules. The active site is located in the core region whereas the peripheral elements of many group I introns contain other elements, such as protein coding sequences and repetitive sequences. While some introns are self-splicing, others depend on protein factors to maintain the correct structure (37). Splicing occurs by two coupled transesterification reactions involving a guanosine cofactor in the first step and separated by a conformational change of the intronic RNA. Self-splicing by group I introns is catalyzed by Mg²⁺ ions, which coordinate directly to the chemically active RNA groups. In the first step of splicing, transesterification occurs when the 3’-OH of an exogenous guanosine attacks the 5’ phosphate of the first nucleotide of the intron (splice site). After the first step, this guanosine is covalently bound to the free 5’ end of the intron. In the second splicing step the 3’ OH group of the free 5’ exon attacks the 3’ splice site, resulting in the ligation of the 5’ and 3’ exons and the release of the intron (38, 39). An exception to this splicing mechanism was observed in an intron located in the 23S rRNA gene of C. burnetii, since it possesses a unique 3’-terminal adenine in place of a conserved guanine (40).

Group II introns are large ribozymes mostly found in bacteria and lower eukaryotes. They are grouped into three major classes based on RNA sequence and structure: IIA, IIB, and IIC (41, 42). Although group II introns are divergent in primary sequence, they fold into highly conserved structures with a six-domain secondary structure formed around a central core region. Of these six domains, Domain V harbors the catalytic active site, where 34 highly conserved nucleotides form a hairpin with a GNRA tetraloop and a 2 nucleotide bulge on the 3’ side (43). In addition, Domain V and Domain I are responsible for binding the two metal ions essential for catalytic activity. Biochemical and genetic studies indicate that some group II introns require an intron-encoded protein to be active for splicing (44, 45). Almost 30 years ago, Steitz and Steitz (46) proposed a two-metal-ion splicing mechanism involving two phosphoryl transfer reactions, which is consistent with more recent observations and proposed mechanisms (47). In this mechanism, first one metal ion stabilizes the 3’ OH of the last 5’ exon nucleotide, the leaving group, while the second metal ion activates the 2’ OH of the branch point adenosine before it leaves the active site. Exon ligation, the second phosphoryl transfer reaction, involves the first metal activating the 3’ OH of the 5’ exon while the second metal stabilizes the 3’ OH of the end of the intron (48). Figure 1C illustrates the catalytic core of a group II intron. Depending on the nucleophile used, the products are different. If the nucleophile is the 2-OH of the branch-site adenosine, the product is a lariat intron, whereas if a water acts as the nucleophile, a linear intron is the product. Not surprisingly, the transesterification reactions can also work in the reverse direction, leading to ligation or ‘reverse splicing’ (47).

In many eukaryotic cells, intron splicing is performed by the spliceosome, a large ribonucleoprotein complex. Unlike many large protein complexes, the spliceosome is not preassembled, but instead it is formed on its substrate from 5 small nuclear RNAs (snRNAs) and approximately 100 proteins (49). These proteins play essential roles and are responsible for the orchestration of the splicing reaction, which involves many different steps. The splicing mechanism can be described in four stages: spliceosome assembly, activation, catalysis, and disassembly. Splicing involves two transesterification reactions, both of which are catalyzed by a single RNA-based active site (48). During catalysis, the needed RNA elements for the reaction are successively delivered into the active site. The spliceosome is qualitatively more sophisticated compared to group II intron and the single biggest difference is the essential role of the many protein components in the spliceosome.

Recent cryo-electron microscopy structures of the spliceosome RNA core have allowed comparison with group II intron crystal structures (50, 51). From these structures it is possible to draw parallels between the RNA active sites, substrate positioning, and product formation in these two intron splicing systems. The comparisons make it clear that the spliceosome and group II introns employ the same chemical mechanism, which should not be surprising given that group II introns and the eukaryotic spliceosome have a common evolutionary origin (52, 53).

Finally, lariat-capping (LC) ribozymes, were originally considered related to the group I ribozyme family (54), but it is now clear that they form a different splicing group (55). LC ribozymes are found downstream of a pre-mRNA coding for a homing endonuclease. After splicing, the mRNA is protected not by a conventional mRNA cap, but by a very small lariat, a product of the branching reaction. Although LC ribozymes have sequence similarities to group I introns, they form lariats as part of the reaction, like group II introns. Structural studies have established that LC ribozymes are distinct from group I introns, although they may have a common evolutionary origin (55).

Conclusions

RNAs play diverse cellular roles and new functions are still being discovered. Furthermore, in recent years, the use of bioinformatics has significantly increased the number of examples of catalytic RNA motifs and identified new ribozymes. To achieve their function, ribozymes fold into their active catalytic conformation forming complicated structures and, in many instances, aided by proteins. Many different reactions catalyzed by natural ribozymes have been uncovered, with most of them involved in phosphoryl transfer reactions (transesterification or hydrolysis). Even in the cases where ribozymes are part of a ribonucleoprotein complex, the RNA component is solely responsible for catalysis. Not surprisingly, there are clear evolutionary relationships between simpler ribozymes and the catalytic core of more elaborated ribonucleoprotein complexes. Although the catalytic mechanism of many ribozymes is now understood, it is likely that our understanding of the versatility of catalytic RNAs will keep growing as more ribozymes are discovered.

Perspectives.

• Catalytic RNAs are central to many biological processes, including translation and splicing.

• The catalytic mechanism of many of these ribozymes is starting to emerge and shows that the RNA, either alone or in complex with proteins, is responsible for catalysis, even when proteins aid the process.

• It is likely that more ribozymes and regulatory RNA molecules will be discovered and that our understanding of the extent of the role of RNA in biology will continue to expand.