Varkud Satellite Ribozyme Research Articles

Catalytic cleave of an RNA substrate that bypasses the reorganization of its secondary structure during substrate recognition by a trans-acting VS ribozyme

Varkud satellite ribozyme (VS ribozyme) is a class of catalytic RNA with self-cleavage activity. The wild-type VS ribozyme has structural modularity with a relatively large catalytic module (H2–H6 elements) and a small substrate module (H1 element). The two modules can be dissected physically, and the substrate H1 RNA is recognized and then cleaved by the rest of the parent ribozyme serving as catalytic RNA. We characterized the catalytic properties of a bimolecular VS ribozyme developed and employed for an in-droplet evolution experiment of the VS ribozyme. We examined the effects of polyamines and several divalent metal ions. The results obtained in this study would be useful for the optimization of laboratory evolution of the VS ribozyme.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1–5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6–8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme.

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.

The Varkud Satellite Ribozyme: A Thirty-Year Journey through Biochemistry, Crystallography, and Computation.

The discovery of catalytic RNAs or ribozymes introduced a new class of enzymes to biology. In addition to their increasingly important roles in modern life, ribozymes are key players in the RNA World hypothesis, which posits that life started or flourished with RNA supporting both genetic and enzymatic functions. Therefore, investigations into the mechanisms of ribozyme function provide an exciting opportunity to examine the foundational principles of biological catalysis. Ribozymes are also attractive model systems to investigate the relationship between structure and function in RNA. Endonucleolytic ribozymes represent the largest class of catalytic RNA, of which the Varkud satellite (VS) ribozyme is structurally the most complex. The last ribozyme to be discovered by accident, the VS ribozyme had eluded structural determination for over two decades. When we solved the first crystal structures of the VS ribozyme, an extensive body of biochemical and biophysical data had accumulated over the years with which we could evaluate the functional relevance of the structure. Conversely, the structures provided a new perspective from which to reexamine the functional data and test new hypotheses. The VS ribozyme is organized in a modular fashion where independently folding domains assemble into the active conformation of the ribozyme via three-way junctions. Structures of the VS ribozyme in complex with its substrate at different stages of activation enabled us to map the structural reorganization of the substrate that must precede catalysis. In addition to defining the global architecture of the RNA, the essential interactions between the substrate and catalytic domains, and the rearrangements in the substrate prior to catalysis, these structures provided detailed snapshots of the ribozyme active site, revealing potential catalytic interactions. High resolution structures of the active site bolstered the view that the catalytic mechanism involved nucleobase-mediated general acid-base catalysis and uncovered additional catalytic interactions between the cleavage site and catalytic residues. Informed by the crystal structures of the VS ribozyme, an integrated experimental and computational approach identified the key players and essential interactions that define the active site of the ribozyme. This confluence of biochemical, structural, and computational studies revealed the catalytic mechanism of the ribozyme at unprecedented detail. Additionally, comparative analyses of the active site structures of the VS ribozyme and other nucleic acid-based endoribonucleases revealed common architectural motifs and strikingly similar catalytic strategies. In this Account, we document the progress of VS ribozyme research starting from its discovery and extending to the elucidation of its detailed catalytic mechanism 30 years later.

In Vitro Selection of Varkud Satellite Ribozyme Variants that Cleave a Modified Stem-Loop Substrate.

In vitro selection is an established approach to create artificial ribozymes with defined activities or to modify the properties of naturally occurring ribozymes. For the Varkud satellite ribozyme of Neurospora, an in vitro selection protocol based on its phosphodiester bond cleavage activity has not been previously reported. Here, we describe a simple protocol for cleavage-based in vitro selection that we recently used to identify variants of the Varkud satellite ribozyme able to target and cleave a non-natural stem-loop substrate derived from the HIV-1 TAR RNA. It allows quick selection of active ribozyme variants from the transcription reaction based on the size of the self-cleavage product without the need for RNA labeling. This results in a streamlined procedure that is easily adaptable to engineer ribozymes with new activities.

Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme

The Varkud Satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free energy relationship measurements with molecular dynamics, molecular solvation theory, and ab initio quantum mechanical/molecular mechanical free energy simulations to gain insight into catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfers in the transition state. Further, we provide evidence for a critical Mg2+ ion in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg2+ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the VS, hairpin, and hammerhead ribozyme classes.

A multi-axial RNA joint with a large range of motion promotes sampling of an active ribozyme conformation.

Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.

Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.

The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.

Mg2+ Binding Promotes SLV as a Scaffold in Varkud Satellite Ribozyme SLI-SLV Kissing Loop Junction

Though the structure of the substrate stem loop I (SLI)-stem loop V (SLV) kissing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dynamics of this Mg2+-dependent loop-loop interaction have been elusive. Specifically, each hairpin loop contains a U-turn motif, but only SLV shows a conformational shift triggered by Mg2+ ion association. Here, we use molecular dynamics simulations to analyze the binding and dynamics of this kissing loop junction. We show that SLV acts as a scaffold, providing stability to the junction. Mg2+ ions associate with SLV when it is part of the junction in a manner similar to when it is unbound, but there is no specificity in Mg2+ binding for the SLI loop. This suggests that the entropic penalty of ordering the larger SLI is too high, allowing SLV to act as a scaffold for multiple substrate loop sequences.

Insights into RNA structure and dynamics from recent NMR and X-ray studies of the Neurospora Varkud satellite ribozyme.

Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS ribozyme recognizes and cleaves its stem‐loop substrate via a mechanism that involves several structural transitions within its stem‐loop substrate. The recent publications of high‐resolution structures of the VS ribozyme, obtained by NMR spectroscopy and X‐ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide‐and‐conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of this divide‐and‐conquer approach reflects the modularity of RNA architecture and the great care taken in identifying the independently‐folding modules. Together with previous biochemical and biophysical characterizations, the recent NMR and X‐ray studies provide a coherent picture into how the VS ribozyme recognizes its stem‐loop substrate. Such in‐depth characterization of this RNA enzyme will serve as a model for future structural and engineering studies of dynamic RNAs and may be particularly useful in planning divide‐and‐conquer investigations. WIREs RNA 2017, 8:e1421. doi: 10.1002/wrna.1421For further resources related to this article, please visit the WIREs website.

Crystal Structure of the Varkud Satellite Ribozyme: Peek into Trans‐active Enzymes of the RNA World

Crystal Structure of the Varkud Satellite Ribozyme: Peek into Trans‐active Enzymes of the RNA World

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5′-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson–Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Crystal structure of the Varkud satellite ribozyme.

Varkud Satellite (VS) ribozyme mediates rolling circle replication of a plasmid found in the Neurospora mitochondria. We report crystal structures of this ribozyme at 3.1Å resolution, revealing an intertwined dimer formed by an exchange of substrate helices. Within each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional significance of active site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.

Nuclear magnetic resonance structure of the III-IV-V three-way junction from the Varkud satellite ribozyme and identification of magnesium-binding sites using paramagnetic relaxation enhancement.

The VS ribozyme is a catalytic RNA found within some natural isolates of Neurospora that is being used as a model system to improve our understanding of RNA structure, catalysis, and engineering. The catalytic domain contains five helical domains (SLII-SLVI) that are organized by two three-way junctions. The III-IV-V junction is required for high-affinity binding of the substrate domain (SLI) through formation of a kissing loop interaction with SLV. Here, we determine the high-resolution nuclear magnetic resonance (NMR) structure of a 47-nucleotide RNA containing the III-IV-V junction (J345). The J345 RNA adopts a Y-shaped fold typical of the family C three-way junctions, with coaxial stacking between stems III and IV and an acute angle between stems III and V. The NMR structure reveals that the core of the III-IV-V junction contains four stacked base triples, a U-turn motif, a cross-strand stacking interaction, an A-minor interaction, and a ribose zipper. In addition, the NMR structure shows that the cCUUGg tetraloop used to stabilize stem IV adopts a novel RNA tetraloop fold, different from the known gCUUGc tetraloop structure. Using Mn(2+)-induced paramagnetic relaxation enhancement, we identify six Mg(2+)-binding sites within J345, including one associated with the cCUUGg tetraloop and two with the junction core. The NMR structure of J345 likely represents the conformation of the III-IV-V junction in the context of the active VS ribozyme and suggests that this junction functions as a dynamic hinge that contributes to substrate recognition and catalysis. Moreover, this study highlights a new role for family C three-way junctions in long-range tertiary interactions.

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

Structural Insights Into Substrate Recognition by the Neurospora Varkud Satellite Ribozyme: Importance of U-Turns at the Kissing-Loop Junction

Substrate recognition by the Neurospora Varkud satellite ribozyme depends on the formation of a magnesium-dependent kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain. From mutagenesis studies, it has been established that this I/V kissing-loop interaction involves three Watson–Crick base pairs and is associated with a structural rearrangement of the SLI substrate that facilitates catalysis. Here, we report the NMR structural characterization of this I/V kissing-loop using isolated stem-loops. NMR studies were performed on different SLI/SLV complexes containing a common SLV and shiftable, preshifted, or double-stranded SLI variants. These studies confirm the presence of three Watson–Crick base pairs at the kissing-loop junction and provide evidence for the structural rearrangement of shiftable SLI variants upon SLV binding. NMR structure determination of an SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitates intermolecular Watson–Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking, help create a continuously stacked structure. These NMR studies provide a structural basis to understand the stability of the I/V kissing-loop interaction and lead us to propose a kinetic model for substrate activation in the VS ribozyme.

Mechanisms of RNA catalysis

Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK(a) = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5' oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2'-O nucleophile by G638 and protonation of the 5'-O leaving group by A756.

Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes

The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg(2+) ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavage activity, a mixture of the two variants leads to cleavage of the A756C RNA, but not the G638A RNA. The rate of cleavage depends on the concentration of the VS G638A RNA, as expected for a bimolecular interaction. This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans.

Fluorine Substituted Adenosines As Probes of Nucleobase Protonation in Functional RNAs

Ionized nucleobases are required for folding, conformational switching, or catalysis in a number of functional RNAs. A common strategy to study these sites employs nucleoside analogues with perturbed pKa, but the interpretation of these studies is often complicated by the chemical modification introduced, in particular modifications that add, remove, or translocate hydrogen bonding groups in addition to perturbing pKa values. In the present study we present a series of fluorine substituted adenosine analogues that produce large changes in N1 pKa values with minimal structural perturbation. These analogues include fluorine for hydrogen substitutions in the adenine ring of adenosine and 7-deaza-adenosine with resulting N1 pKa values spanning more than 4 pKa units. To demonstrate the utility of these analogues we have conducted a nucleotide analogue interference mapping (NAIM) study on a self-ligating construct of the Varkud Satellite (VS) ribozyme. We find that each of the analogues is readily incorporated by T7 RNA polymerase and produces fully active transcripts when substituted at the majority of sites. Strong interferences are observed for three sites known to be critical for VS ribozyme function, most notably A756. Substitutions at A756 lead to slight enhancements in activity for elevated pKa analogues and dramatic interferences in activity for reduced pKa analogues, supporting the proposed catalytic role for this base. The structural similarity of these analogues, combined with their even incorporation and selective interference, provides an improved method for identifying sites of adenosine protonation in a variety of systems.