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.

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.

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.

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.

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.

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.

A relaxed active site after exon ligation by the group I intron.

During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

A technique to enzymatically construct libraries which express short hairpin RNA of arbitrary stem length.

Short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) usually used for RNA interference (RNAi) are double-stranded RNAs (dsRNAs) of 21 base pairs. However, siRNAs and shRNAs of longer stem length have been reported to show more potent gene silencing. Here, we report a new technique to enzymatically construct shRNA libraries containing clones from firefly luciferase cDNA and Jurkat cDNA. The technique allowed the efficacious generation of shRNAs of arbitrary stem length as desired, providing the clones which potently silenced the specified gene expression and presenting a high efficiency rate of gene silencing. Our results indicate that the technique permits the rapid, efficient, and low-cost preparation of genomewide shRNA expression libraries not only for humans and mice but also for sorts of biological species and that the relevant libraries are applicable for the search of genes related to phenotype changes and of new targets for drug discovery.

Structure-function analysis of yeast tRNA ligase.

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages.

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.

The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases.

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion.

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

Yeast tRNA ligase (Trl1) converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO4, 3'-5' phosphodiester at the splice junction. Trl1 performs three reactions: (i) the 2',3'-cyclic phosphate of the proximal fragment is hydrolyzed to a 3'-OH, 2'-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5'-OH of the distal fragment is phosphorylated by an NTP-dependent polynucleotide kinase; and (iii) the 3'-OH, 2'-PO4, and 5'-PO4 ends are sealed by an ATP-dependent RNA ligase. Trl1 consists of an N-terminal adenylyltransferase domain that resembles T4 RNA ligase 1, a central domain that resembles T4 polynucleotide kinase, and a C-terminal CPD domain that resembles the 2H phosphotransferase enzyme superfamily. Here we show that all three domains are essential in vivo, although they need not be linked in the same polypeptide. We identify five amino acids in the adenylyltransferase domain (Lys114, Glu266, Gly267, Lys284, and Lys286) that are essential for Trl1 activity and are located within motifs I (114KANG117), IV (266EGFVI270), and V (282FFKIK286) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligases 1 and 2. Mutations K404A and T405A in the P-loop (401GXGKT405) of the central kinase-like domain had no effect on Trl1 function in vivo. The K404A and T405A mutations eliminated ATP-dependent kinase activity but preserved GTP-dependent kinase activity. A double alanine mutant in the P-loop was lethal in vivo and abolished GTP-dependent kinase activity. These results suggest that GTP is the physiological substrate and that the Trl1 kinase has a single NTP binding site of which the P-loop is a component. Two other mutations in the central domain were lethal in vivo and either abolished (D425A) or severely reduced (R511A) GTP-dependent RNA kinase activity in vitro. Mutations of the signature histidines of the CPD domain were either lethal (H777A) or conferred a ts growth phenotype (H673A).

Kinetoplastid RNA editing ligases: complex association, characterization, and substrate requirements.

RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.