Ribozyme Domain Research Articles

Low prevalence of hepatitis delta infection in Cuban HBsAg carriers: Prospect for elimination.

Infection with hepatitis delta virus (HDV) is one of the most severe hepatitis B virus (HBV) complications, with a more rapid progression to cirrhosis and an increased risk of hepatic decompensation and death. Data on HDV infection in Cuba are limited. The aims of our study were to determine the HDV prevalence in HBsAg carriers and to characterize the HDV strains circulating. The data were used to assess the possibility of HDV elimination in the Cuban HBV epidemiological setting. Five hundred and two serum samples from the same number of HBsAg carriers collected in the period 2006-2019 from all over the country were tested for anti-HDV total antibodies. If positive, the samples were analyzed for HDV-RNA using Real-Time RT-PCR targeting the ribozyme and HD antigen domains followed by genotyping based on phylogenetic analysis. Two samples were anti-HDV positive [0.39% (95% CI 0.11-1.44)]. One of them was also HDV-RNA positive. Clinically, the patient with active HDV infection had compensated liver cirrhosis. Phylogenetic analysis showed that the virus belonged to genotype 1 and thus clustered with contemporary strains from North America, Europe, Middle East, and Asia. This is the first HDV study, including molecular detection and virus characterization, done after the introduction of the universal childhood anti-hepatitis B vaccination. The very low prevalence of HDV infection in HBsAg carriers combined with the high HBV vaccination coverage of all newborn children, of previously identified risk groups, and of the general population currently under 40 years of age suggests that HDV elimination is feasible in Cuba if the success in HBV control is maintained.

Unprecedented frequency of mitochondrial introns in colonial bilaterians

Animal mitogenomes are typically devoid of introns. Here, we report the largest number of mitochondrial introns ever recorded from bilaterian animals. Mitochondrial introns were identified for the first time from the phylum Bryozoa. They were found in four species from three families (Order Cheilostomatida). A total of eight introns were found in the complete mitogenome of Exechonella vieirai, and five, 17 and 18 introns were found in the partial mitogenomes of Parantropora penelope, Discoporella cookae and Cupuladria biporosa, respectively. Intron-encoded protein domains reverse transcriptase and intron maturase (RVT-IM) were identified in all species. Introns in E. vieirai and P. penelope had conserved Group II intron ribozyme domains V and VI. Conserved domains were lacking from introns in D. cookae and C. biporosa, preventing their further categorization. Putative origins of metazoan introns were explored in a phylogenetic context, using an up-to-date alignment of mitochondrial RVT-IM domains. Results confirmed previous findings of multiple origins of annelid, placozoan and sponge RVT-IM domains and provided evidence for common intron donor sources across metazoan phyla. Our results corroborate growing evidence that some metazoans with regenerative abilities (i.e. placozoans, sponges, annelids and bryozoans) are susceptible to intron integration, most likely via horizontal gene transfer.

Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets.

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains.

Insight into the Contribution and Disruption of Host Processes during HDV Replication.

Hepatitis delta virus (HDV) is unique among animal viruses. HDV is a satellite virus of the hepatitis B virus (HBV), however it shares no sequence similarity with its helper virus and replicates independently in infected cells. HDV is the smallest human pathogenic RNA virus and shares numerous characteristics with viroids. Like viroids, HDV has a circular RNA genome which adopts a rod-like secondary structure, possesses ribozyme domains, replicates in the nucleus of infected cells by redirecting host DNA-dependent RNA polymerases (RNAP), and relies heavily on host proteins for its replication due to its small size and limited protein coding capacity. These similarities suggest an evolutionary relationship between HDV and viroids, and information on HDV could allow a better understanding of viroids and might globally help understanding the pathogenesis and molecular biology of these subviral RNAs. In this review, we discuss the host involvement in HDV replication and its implication for HDV pathogenesis.

Mutational Analysis of Eggplant Latent Viroid RNA Circularization by the Eggplant tRNA Ligase in Escherichia coli.

Eggplant latent viroid (ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants (Solanum melongena L.). Like other viroid species that belong to the family Avsunviroidae, ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5′-hydroxyl and 2′,3′-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a quasi-rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd quasi-double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in Escherichia coli, they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates.

RNA structure inference through chemical mapping after accidental or intentional mutations

Despite the critical roles RNA structures play in regulating gene expression, sequencing-based methods for experimentally determining RNA base pairs have remained inaccurate. Here, we describe a multidimensional chemical-mapping method called "mutate-and-map read out through next-generation sequencing" (M2-seq) that takes advantage of sparsely mutated nucleotides to induce structural perturbations at partner nucleotides and then detects these events through dimethyl sulfate (DMS) probing and mutational profiling. In special cases, fortuitous errors introduced during DNA template preparation and RNA transcription are sufficient to give M2-seq helix signatures; these signals were previously overlooked or mistaken for correlated double-DMS events. When mutations are enhanced through error-prone PCR, in vitro M2-seq experimentally resolves 33 of 68 helices in diverse structured RNAs including ribozyme domains, riboswitch aptamers, and viral RNA domains with a single false positive. These inferences do not require energy minimization algorithms and can be made by either direct visual inspection or by a neural-network-inspired algorithm called M2-net. Measurements on the P4-P6 domain of the Tetrahymena group I ribozyme embedded in Xenopus egg extract demonstrate the ability of M2-seq to detect RNA helices in a complex biological environment.

Rational Engineering of a Modular Group I Ribozyme to Control Its Activity by Self-Dimerization.

We designed and constructed a dimer of the Tetrahymena group I ribozyme the activity of which is regulated by self-dimerization. This dimer was rationally designed by utilizing the P5abc and ΔP5abc domains as large RNA motifs. This strategy enabled us to install large ribozyme functions into an RNA structure. This is a step toward expanding the field of RNA nanotechnology beyond the limitation of using only relatively small functional motifs. Self-dimerization can also be rationally programmed by modular engineering of RNA interaction motifs. In this chapter, we present the procedure for the rational design and construction of large ribozyme domains based on RNA tertiary structures. We also describe the electrophoresis mobility shift assay (EMS) and several ribozyme activity assays to confirm the ribozyme function and its regulation. We have succeeded in construction of tecto-GIRz.

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity.

RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP) that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP) RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron

When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled.

RNA World research-still evolving.

RNA World research-still evolving.

Molecular characterization of a new member of the lariat capping twin-ribozyme introns.

BackgroundTwin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Naegleria amoeboflagellates and the myxomycete Didymium iridis.ResultsWe characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents an efficient self-cleaving ribozyme that generates a small 2′,5′ lariat cap at the 5′ end of the homing endonuclease mRNA, and thus contributes to intron mobility.ConclusionsThe discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a putative regulatory RNA element (AP2.1) in the Allovahlkampfia LC ribozyme that involves homing endonuclease mRNA coding sequences as an important structural component.

Ribozyme-independent replication of a defective hepatitis D virus RNA derived from hepatitis B/D patients receiving antiviral therapy

Novel hepatitis D virus (HDV) RNA mutants carrying large fragment deletions were identified in the serum samples of two hepatitis B/D patients receiving antiviral therapy. Sequence analysis revealed that the deleted regions encompassed both ribozyme domains. The mutant persisted in the serum samples for at least 2 and 10months, respectively in the two patients, raising the question of whether such mutants could replicate in the absence of ribozyme domains. Thirty anti-HDV antibody-positive serum samples derived from 17 patients receiving antiviral therapy were submitted for RT-PCR detection of HDV RNA deletion mutants. Large fragment HDV RNA deletions were found in 4 patients. Of them, two had liver biopsy samples available. Northern blot analysis revealed high molecular weight HDV RNA replication intermediates, genomic and anti-genomic senses, in the liver tissues. Transfection of an in vitro transcribed HDV RNA deletion mutant (1.28kb) into Huh7 and HepG2 cells also resulted in generation of high molecular weight HDV RNA species in the hepatoma cells (>6.5kb) with secretion of a 6.5kb HDV RNA species into the medium. In conclusion, we discovered novel large fragment deletion mutants of HDV RNA in hepatitis B/D patients receiving antiviral therapy. Such mutants did not contain ribozyme domains but could replicate in the liver cells to generate high molecular weight but not unit-length HDV RNA.

Comprehensive Phylogenetic Analysis of Bacterial Group II Intron-Encoded ORFs Lacking the DNA Endonuclease Domain Reveals New Varieties

Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP) ORF within the ribozyme domain IV (DIV). This ORF encodes an N-terminal reverse transcriptase (RT) domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D) region followed by a DNA endonuclease (En) domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns). On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.

Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

BackgroundGroup II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.ResultsIn this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.ConclusionsThe excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37 degrees C and at a concentration of MgCl(2) as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 3' OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.

ChemInform Abstract: Synthesis of 4-Thiothymine Based Photolabels as New Tools for Nucleic Acids Structural Studies in Solution: Formation of Long-Range Photo-Cross-Links within a Hammerhead Ribozyme Domain.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Investigating a New Generation of Ribozymes in Order to Target HCV

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity “on” solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (−) strands.

Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.

Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron

BackgroundRibosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA.ResultsTo elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus.ConclusionThe Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%), leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.