Reverse Genetics Reveals a Role of Rotavirus VP3 Phosphodiesterase Activity in Inhibiting RNase L Signaling and Contributing to Intestinal Viral Replication In Vivo.

Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo Here, we present both in vitro and in vivo evidence that the host interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (OAS) and RNase L pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2'-5'-phosphodiesterase (PDE) domain to counteract RNase L-mediated antiviral signaling. Using an RV reverse-genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at low levels in the small intestine and were shed less in the feces of wild-type mice, and such defects were rescued in Rnasel-/- suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well-characterized function as the viral RNA-capping enzyme.IMPORTANCE Rotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low- to middle-income countries, where the burden of the diseases is the most severe. With the ultimate goal of improving current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus-encoded VP3 antagonizes RNase L activity both in vitro and in vivo These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for next-generation rotavirus vaccine design.

tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.

Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation.

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, and , the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in , but not in , despite the absence of a substrate-binding site in . Loop-loop interactions of - and -variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.

Structure and two-metal mechanism of fungal tRNA ligase.

Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central GTP-dependent polynucleotide kinase (KIN) domains that heal the broken ends to generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain (LIG). Here we report crystal structures of the Trl1-LIG domain from Chaetomium thermophilum at two discrete steps along the reaction pathway: the covalent LIG-(lysyl-Nζ)-AMP•Mn2+ intermediate and a LIG•ATP•(Mn2+)2 Michaelis complex. The structures highlight a two-metal mechanism whereby a penta-hydrated metal complex stabilizes the transition state of the ATP α phosphate and a second metal bridges the ß and γ phosphates to help orient the pyrophosphate leaving group. A LIG-bound sulfate anion is a plausible mimetic of the essential RNA terminal 2'-PO4. Trl1-LIG has a distinctive C-terminal domain that instates fungal Trl1 as the founder of an Rnl6 clade of ATP-dependent RNA ligase. We discuss how the Trl1-LIG structure rationalizes the large body of in vivo structure-function data for Saccharomyces cerevisiae Trl1.

Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

Real-Time Detection of a Self-Replicating RNA Enzyme.

A system was developed to detect the self-replication of an RNA enzyme in real time. The enzyme is an RNA ligase that undergoes exponential amplification at a constant temperature and can be made to operate in a ligand-dependent manner. The real-time system is based on a fluorimetric readout that directly couples the ligation event to an increase in florescence signal that can be monitored using standard instrumentation. The real-time system can also operate entirely with l-RNA, which is not susceptible to degradation by ribonucleases that are present in biological samples. The system is analogous to real-time PCR, but with the potential to detect small molecules, proteins, and other targets that can be recognized by a suitable aptamer. The ligand-dependent self-replication of RNA has potential applications in molecular diagnostics and biosensing that benefit from the rapid, precise, and real-time detection of various target molecules.

Crystal structure of a DNA catalyst.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.

Kinetic properties of an RNA enzyme that undergoes self-sustained exponential amplification.

A special class of biochemical reactions involves a set of enzymes that generate additional copies of themselves and transfer heritable information from parent to progeny molecules, thus providing the basis for genetics and Darwinian evolution. Such a process has been realized with a pair of self-replicating RNA enzymes that undergo exponential amplification at a constant temperature. Exponential growth requires that the rate of production of new enzymes be directly proportional to the existing concentration of enzymes, which is the case for this system and provides a doubling time of ~20 min. However, the catalytic rate of the underlying enzymes is ~100-fold faster than the observed rate of replication. As in biological replication, other aspects of the system limit the generation time, chiefly the propensity of the substrate molecules to form nonproductive complexes that limit their availability for replication. An analysis of this and other kinetic properties of the self-replicating RNA enzymes reveals how exponential amplification is achieved and how the rate of amplification might be increased.

Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH.

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2',3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.

GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus.

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment.

All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.

RNA labeling, conjugation and ligation.

Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA either directly or indirectly through post-synthetic labeling methodologies that have enabled a broader understanding of RNA structure and function. Readily applicable modifications to RNA can range from isotopic labels and fluorescent or other molecular probes to protein, lipid, glycoside or nucleic acid conjugates that can be introduced using combinations of synthetic chemistry, enzymatic incorporation and various conjugation chemistries. These labels, conjugations and ligations to RNA are quintessential for further investigation and applications of RNA as they enable the visualization, structural elucidation, localization, and biodistribution of modified RNA.

Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

Closing the circle: replicating RNA with RNA.

How life emerged on this planet is one of the most important and fundamental questions of science. Although nearly all details concerning our origins have been lost in the depths of time, there is compelling evidence to suggest that the earliest life might have exploited the catalytic and self-recognition properties of RNA to survive. If an RNA based replicating system could be constructed in the laboratory, it would be much easier to understand the challenges associated with the very earliest steps in evolution and provide important insight into the establishment of the complex metabolic systems that now dominate this planet. Recent progress into the selection and characterization of ribozymes that promote nucleotide synthesis and RNA polymerization are discussed and outstanding problems in the field of RNA-mediated RNA replication are summarized.

Crystal structure of the catalytic core of an RNA-polymerase ribozyme.

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

Evolution in an RNA world.

A long-standing research goal has been to develop a self-sustained chemical system that is capable of undergoing Darwinian evolution. The notion of primitive RNA-based life suggests that this goal might be achieved by constructing an RNA enzyme that catalyzes the replication of RNA molecules, including the RNA enzyme itself. This reaction was demonstrated recently in a cross-catalytic system involving two RNA enzymes that catalyze each other's synthesis from a total of four component substrates. The cross-replicating RNA enzymes undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of approximately 1 hour and can be continued indefinitely. Small populations of cross-replicating RNA enzymes can be made to compete for limited resources within a common environment. The molecules reproduce with high fidelity but occasionally give rise to recombinants that also can replicate. Over the course of many "generations" of selective amplification, novel variants arise and grow to dominate the population based on their relative fitness under the chosen reaction conditions. This is the first example, outside of biology, of evolutionary adaptation in a molecular genetic system.

Autocatalytic aptazymes enable ligand-dependent exponential amplification of RNA.

RNA enzymes have been developed that undergo self-sustained replication at a constant temperature in the absence of proteins. These RNA molecules amplify exponentially through a cross-replicative process, whereby two enzymes catalyze each other's synthesis by joining component oligonucleotides. Other RNA enzymes have been made to operate in a ligand-dependent manner by combining a catalytic domain with a ligand-binding domain (aptamer) to produce an 'aptazyme'. The principle of ligand-dependent RNA catalysis has now been extended to the cross-replicating RNA enzymes so that exponential amplification occurs in the presence, but not the absence, of the cognate ligand. The exponential growth rate of the RNA depends on the concentration of the ligand, allowing one to determine the concentration of ligand in a sample. This process is analogous to quantitative PCR (qPCR) but can be generalized to a wide variety of targets, including proteins and small molecules that are relevant to medical diagnostics and environmental monitoring.

Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus.

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed approximately 27% identity to that of the 2'-5' tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2',3'-cyclic phosphate and 5'-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPgammaS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein.

Self-sustained replication of an RNA enzyme.

An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates. These cross-replicating RNA enzymes undergo self-sustained exponential amplification in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about 1 hour and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.

Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo.

Yeast and human Clp1 proteins are homologous components of the mRNA 3'-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5'-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5'-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Delta strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5'-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.