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Sample records for synthetic splicing ribozymes

  1. Trans-splicing with the group I intron ribozyme from Azoarcus

    PubMed Central

    Dolan, Gregory F.; Müller, Ulrich F.

    2014-01-01

    Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNAIle group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5′ terminus. In contrast to the previously optimized 5′-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5′ portion and the ribozyme 3′ exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme. PMID:24344321

  2. Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

    PubMed

    Byun, Jonghoe; Lan, Ning; Long, Meredith; Sullenger, Bruce A

    2003-10-01

    Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

  3. RNA-based networks: using RNA aptamers and ribozymes as synthetic genetic devices.

    PubMed

    Weigand, Julia E; Wittmann, Alexander; Suess, Beatrix

    2012-01-01

    Within the last few years, a set of synthetic riboswitches has been engineered, which expands the toolbox of genetic regulatory devices. Small molecule binding aptamers have been used for the design of such riboswitches by insertion into untranslated regions of mRNAs, exploiting the fact that upon ligand binding the RNA structure interferes either with translation initiation or pre-mRNA splicing in yeast. In combination with self-cleaving ribozymes, aptamers have been used to modulate RNA stability. In this chapter, we discuss the applicability of different aptamers, ways to identify novel genetic devices, the pros and cons of various insertion sites and the application of allosteric ribozymes. Our expertise help to apply synthetic riboswitches to engineer complex genetic circuits. PMID:22083741

  4. Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

    PubMed Central

    Curtis, Edward A.; Bartel, David P.

    2013-01-01

    The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 1014 different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties. PMID:23798664

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

    PubMed

    Monachello, Dario; Michel, François; Costa, Maria

    2016-03-01

    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.

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

    PubMed

    Monachello, Dario; Michel, François; Costa, Maria

    2016-03-01

    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. PMID:26769855

  7. Who Activates the Nucleophile in Ribozyme Catalysis? An Answer from the Splicing Mechanism of Group II Introns.

    PubMed

    Casalino, Lorenzo; Palermo, Giulia; Rothlisberger, Ursula; Magistrato, Alessandra

    2016-08-24

    Group II introns are Mg(2+)-dependent ribozymes that are considered to be the evolutionary ancestors of the eukaryotic spliceosome, thus representing an ideal model system to understand the mechanism of conversion of premature messenger RNA (mRNA) into mature mRNA. Neither in splicing nor for self-cleaving ribozymes has the role of the two Mg(2+) ions been established, and even the way the nucleophile is activated is still controversial. Here we employed hybrid quantum-classical QM(Car-Parrinello)/MM molecular dynamics simulations in combination with thermodynamic integration to characterize the molecular mechanism of the first and rate-determining step of the splicing process (i.e., the cleavage of the 5'-exon) catalyzed by group II intron ribozymes. Remarkably, our results show a new RNA-specific dissociative mechanism in which the bulk water accepts the nucleophile's proton during its attack on the scissile phosphate. The process occurs in a single step with no Mg(2+) ion activating the nucleophile, at odds with nucleases enzymes. We suggest that the novel reaction path elucidated here might be an evolutionary ancestor of the more efficient two-metal-ion mechanism found in enzymes. PMID:27309711

  8. Ribozyme-based insulator parts buffer synthetic circuits from genetic context

    PubMed Central

    Lou, Chunbo; Stanton, Brynne; Chen, Ying-Ja; Munsky, Brian; Voigt, Christopher A

    2014-01-01

    Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression1–4. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit5–9. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as ‘insulator parts’, is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs. PMID:23034349

  9. SASD: the Synthetic Alternative Splicing Database for identifying novel isoform from proteomics

    PubMed Central

    2013-01-01

    Background Alternative splicing is an important and widespread mechanism for generating protein diversity and regulating protein expression. High-throughput identification and analysis of alternative splicing in the protein level has more advantages than in the mRNA level. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, based on the peptidomic database of human protein isoforms for proteomics experiments, our objective is to design a new alternative splicing database to 1) provide more coverage of genes, transcripts and alternative splicing, 2) exclusively focus on the alternative splicing, and 3) perform context-specific alternative splicing analysis. Results We used a three-step pipeline to create a synthetic alternative splicing database (SASD) to identify novel alternative splicing isoforms and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. First, we extracted information on gene structures of all genes in the Ensembl Genes 71 database and incorporated the Integrated Pathway Analysis Database. Then, we compiled artificial splicing transcripts. Lastly, we translated the artificial transcripts into alternative splicing peptides. The SASD is a comprehensive database containing 56,630 genes (Ensembl gene IDs), 95,260 transcripts (Ensembl transcript IDs), and 11,919,779 Alternative Splicing peptides, and also covering about 1,956 pathways, 6,704 diseases, 5,615 drugs, and 52 organs. The database has a web-based user interface that allows users to search, display and download a single gene/transcript/protein, custom gene set, pathway, disease, drug, organ related alternative splicing. Moreover, the quality of the database was validated with comparison to other

  10. Spliced synthetic genes as internal controls in RNA sequencing experiments.

    PubMed

    Hardwick, Simon A; Chen, Wendy Y; Wong, Ted; Deveson, Ira W; Blackburn, James; Andersen, Stacey B; Nielsen, Lars K; Mattick, John S; Mercer, Tim R

    2016-09-01

    RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNA-seq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome. PMID:27502218

  11. Synthetic mRNA splicing modulator compounds with in vivo antitumor activity.

    PubMed

    Lagisetti, Chandraiah; Pourpak, Alan; Goronga, Tinopiwa; Jiang, Qin; Cui, Xiaoli; Hyle, Judith; Lahti, Jill M; Morris, Stephan W; Webb, Thomas R

    2009-11-26

    We report our progress on the development of new synthetic anticancer lead compounds that modulate the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and carbamate analogues. Further, we describe initial animal studies demonstrating the antitumor efficacy of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a new 1,3-dioxane series of active analogues. We confirm that compound 5 inhibits the splicing of mRNA in cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of these compounds as potential human therapeutics.

  12. Ribozymes and their medical implications

    SciTech Connect

    Cech, T.R.

    1988-11-25

    Certain RNA molecules can mediate their own cleavage or splicing or act as enzymes to promote reactions on substrate RNA molecules. Thus, RNA is not restricted to being a passive carrier of genetic information but can have an active role in directing cellular biochemistry. These findings suggest the possibility that other cellular RNA's, including the RNA components of small nuclear ribonucleoproteins, of the ribosome, and of various ribonucleoprotein enzymes, are catalysts. RNA enzymes (ribozymes) can be used as sequence-specific RNA cleavage agents in vitro, providing useful tools for biochemical studies of RNA. On a more speculative note, ribozymes directed against viral RNAs have the potential of serving as therapeutic agents. Finally, some infectious agents including hepatitis delta virus and perhaps poliovirus and rhinoviruses, are themselves ribozymes, providing potential targets for pharmaceuticals.

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

    PubMed Central

    2014-01-01

    Background Twin-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. Results We 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. Conclusions The 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. PMID:25342998

  14. Mechanism for gene control by a natural allosteric group I ribozyme.

    PubMed

    Chen, Andy G Y; Sudarsan, Narasimhan; Breaker, Ronald R

    2011-11-01

    An allosteric ribozyme consisting of a metabolite-sensing riboswitch and a group I self-splicing ribozyme was recently found in the pathogenic bacterium Clostridium difficile. The riboswitch senses the bacterial second messenger c-di-GMP, thereby controlling 5'-splice site choice by the downstream ribozyme. The proximity of this allosteric ribozyme to the open reading frame (ORF) for CD3246 suggests that coenzyme-mediated regulation of splicing controls expression of this putative virulence gene. In the presence of c-di-GMP, the allosteric ribozyme in the CD3246 precursor transcript generates a spliced transcript that retains the riboswitch aptamer. In the absence of c-di-GMP, the ribozyme mediates an alternative GTP attack that results in a truncated transcript (alternative GTP-attack product). Using reporter assays in Escherichia coli, we investigated the difference in gene expression between the spliced product and the alternative GTP-attack product. We provide evidence that CD3246 gene expression is activated if allosteric ribozyme splicing creates a ribosome binding site (RBS) for translation from a UUG start codon. In addition, biochemical and genetic analyses reveal that the riboswitch may further control CD3246 expression by revealing or occluding this newly formed RBS. Therefore, this architecture provides the riboswitch with a mechanism for extended regulation after splicing has occurred or as a backup mechanism for suppression of translation in the event of misregulated splicing.

  15. Inhibition of pre-mRNA splicing by a synthetic Blom7α-interacting small RNA.

    PubMed

    Löscher, Marlies; Schosserer, Markus; Dausse, Eric; Lee, Kiseok; Ajuh, Paul; Grillari-Voglauer, Regina; Lamond, Angus I; Toulmé, Jean-Jacques; Grillari, Johannes

    2012-01-01

    Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEV(Prp19/Pso4), an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)(1-4) C(2-6) (U/A)(1-5), we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development. PMID:23144703

  16. Identification of the nucleotides in the A-rich bulge of the Tetrahymena ribozyme responsible for an efficient self-splicing reaction.

    PubMed

    Ikawa, Y; Okada, A; Imahori, H; Shiraishi, H; Inoue, T

    1997-10-01

    P5abc is a large extension of the P5 element characteristic of subclasses IC1 and IC2 of group I introns. It has a conserved region termed the A-rich bulge, that is responsible for activation of the Tetrahymena self-splicing intron. By employing a modified color-colony assay system, we identified four adenosines in the bulge that are responsible for an efficient splicing reaction. On comparison with the X-ray crystal structure of the P4-5-6 domains of the Tetrahymena intron, three adenosines at positions 183, 184, and 186 were found to be identical to those significantly contributing to the formation of its tertiary structure. However, our results show that an adenosine at 187 is involved in the formation of a Watson-Crick base pair with U135, although it forms a Hoogsteen base pair in the crystal structure. PMID:9399595

  17. Ribozyme-Spherical Nucleic Acids

    PubMed Central

    Hao, Liangliang; Kouri, Fotini M.; Briley, William E.; Stegh, Alexander H.; Mirkin, Chad A.

    2015-01-01

    Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant RNA such as small interfering RNA (siRNA) and microRNA (miRNA). Herein, a synthetic strategy that makes use of the spherical nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported. The properties of this novel ribozyme SNA are characterized in the context of the targeted knockdown of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein involved in chemotherapeutic resistance of solid tumors, foremost glioblastoma multiforme (GBM). Data showing the direct cleavage of full-length MGMT mRNA, knockdown of MGMT protein, and increased sensitization of GBM cells to therapy-mediated apoptosis, independent of transfection agents, provide compelling evidence for the promising properties of this new chemical architecture. PMID:26271335

  18. A versatile cis-blocking and trans-activation strategy for ribozyme characterization

    PubMed Central

    Kennedy, Andrew B.; Liang, Joe C.; Smolke, Christina D.

    2013-01-01

    Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices. PMID:23155065

  19. Twister ribozymes as highly versatile expression platforms for artificial riboswitches

    PubMed Central

    Felletti, Michele; Stifel, Julia; Wurmthaler, Lena A.; Geiger, Sophie; Hartig, Jörg S.

    2016-01-01

    The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class. PMID:27670347

  20. The unforeseeable hammerhead ribozyme

    PubMed Central

    Hammann, Christian

    2009-01-01

    Despite its small size, the complex behavior of the hammerhead ribozyme keeps surprising us, even more than 20 years after its discovery. Here, we summarize recent developments in the field, in particular the discovery of the first split hammerhead ribozyme. PMID:20948624

  1. High-throughput assay and engineering of self-cleaving ribozymes by sequencing

    PubMed Central

    Kobori, Shungo; Nomura, Yoko; Miu, Anh; Yokobayashi, Yohei

    2015-01-01

    Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes. PMID:25829176

  2. A Faster Triphosphorylation Ribozyme

    PubMed Central

    Dolan, Gregory F.; Akoopie, Arvin; Müller, Ulrich F.

    2015-01-01

    In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1. PMID:26545116

  3. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

    NASA Technical Reports Server (NTRS)

    Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

  4. A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

    PubMed Central

    Gleitsman, Kristin R.

    2014-01-01

    Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

  5. The ubiquitous hammerhead ribozyme

    PubMed Central

    Hammann, Christian; Luptak, Andrej; Perreault, Jonathan; de la Peña, Marcos

    2012-01-01

    The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs. PMID:22454536

  6. Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing

    PubMed Central

    Rigo, Frank; Hua, Yimin; Chun, Seung J; Prakash, Thazha P; Krainer, Adrian R; Bennett, C Frank

    2016-01-01

    We describe a new technology for recruiting specific proteins to RNA through selective recognition of heteroduplexes formed with chemically modified antisense oligonucleotides (ASOs). Typically, ASOs function by hybridizing to their RNA targets and blocking the binding of single-stranded RNA–binding proteins. Unexpectedly, we found that ASOs with 2′-deoxy-2′-fluoro (2′-F) nucleotides, but not with other 2′ chemical modifications, have an additional property: they form heteroduplexes with RNA that are specifically recognized by the interleukin enhancer-binding factor 2 and 3 complex (ILF2/3). 2′-F ASO–directed recruitment of ILF2/3 to RNA can be harnessed to control gene expression by modulating alternative splicing of target transcripts. ILF2/3 recruitment to precursor mRNA near an exon results in omission of the exon from the mature mRNA, both in cell culture and in mice. We discuss the possibility of using chemically engineered ASOs that recruit specific proteins to modulate gene expression for therapeutic intervention. PMID:22504300

  7. A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation.

    PubMed Central

    Tuschl, T; Sharp, P A; Bartel, D P

    2001-01-01

    In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation. PMID:11214178

  8. Intronic hammerhead ribozymes in mRNA biogenesis.

    PubMed

    García-Robles, Inmaculada; Sánchez-Navarro, Jesús; de la Peña, Marcos

    2012-11-01

    Small self-cleaving ribozymes are a group of natural RNAs that are capable of catalyzing their own and sequence-specific endonucleolytic cleavage. One of the most studied members is the hammerhead ribozyme (HHR), a catalytic RNA originally discovered in subviral plant pathogens but recently shown to reside in a myriad of genomes along the tree of life. In eukaryotes, most of the genomic HHRs seem to be related to short interspersed retroelements, with the main exception of a group of strikingly conserved ribozymes found in the genomes of all amniotes (reptiles, birds and mammals). These amniota HHRs occur in the introns of a few specific genes, and clearly point to a preserved biological role during pre-mRNA biosynthesis. More specifically, bioinformatic analysis suggests that these intronic ribozymes could offer a new form of splicing regulation of the mRNA of higher vertebrates. We review here the latest advances in the discovery and biological characterization of intronic HHRs of vertebrates, including new conserved examples in the genomes of the primitive turtle and coelacanth fish. PMID:23109545

  9. In vivo screening of ligand-dependent hammerhead ribozymes.

    PubMed

    Saragliadis, Athanasios; Klauser, Benedikt; Hartig, Jörg S

    2012-01-01

    The development of artificial switches of gene expression is of high importance for future applications in biotechnology and synthetic biology. We have developed a powerful RNA-based system which allows for the ligand-dependent and reprogrammable control of gene expression in Escherichia coli. Our system makes use of the hammerhead ribozyme (HHR) which acts as molecular scaffold for the sequestration of the ribosome binding site (RBS), mimicking expression platforms in naturally occurring riboswitches. Aptamer domains can be attached to the ribozyme as exchangeable ligand-sensing modules. Addition of ligands to the bacterial growth medium changes the activity of the ligand-dependent self-cleaving ribozyme which in turn switches gene expression. In this chapter, we describe the in vivo screening procedure allowing for reprogramming the ligand-specificity of our system. PMID:22315086

  10. Small Self-cleaving Ribozymes

    PubMed Central

    Ferré-D'Amaré, Adrian R.; Scott, William G.

    2010-01-01

    Summary The hammerhead, hairpin, hepatitis delta virus (HDV), Varkud Satellite (VS), and glmS ribozymes catalyze sequence-specific intramolecular cleavage of RNA. They range between 50 and 150 nucleotides in length, and are known as the “small self-cleaving ribozymes.” Except for the glmS ribozyme that functions as a riboswitch in Gram-positive bacteria, they were originally discovered as domains of satellite RNAs. However, recent studies show that several of them are broadly distributed in genomes of organisms from many phyla. Each of these ribozymes has a unique overall architecture and active site organization. Crystal structures have revealed how RNA active sites can bind preferentially to the transition state of a reaction, whereas mechanistic studies have shown that nucleobases can efficiently perform general acid–base and electrostatic catalysis. This versatility explains the abundance of ribozymes in contemporary organisms and also supports a role for catalytic RNAs early in evolution. PMID:20843979

  11. Complete RNA inverse folding: computational design of functional hammerhead ribozymes

    PubMed Central

    Dotu, Ivan; Garcia-Martin, Juan Antonio; Slinger, Betty L.; Mechery, Vinodh; Meyer, Michelle M.; Clote, Peter

    2014-01-01

    Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/. PMID:25209235

  12. Ribozymes and Riboswitches: Modulation of RNA Function by Small Molecules†

    PubMed Central

    Zhang, Jinwei; Lau, Matthew W.; Ferré-D'Amaré, Adrian R.

    2010-01-01

    Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule binding-induced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated. PMID:20931966

  13. Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes.

    PubMed

    Beilstein, Kim; Wittmann, Alexander; Grez, Manuel; Suess, Beatrix

    2015-05-15

    Robust synthetic devices are requisite for the construction of synthetic genetic circuits and important scientific and technological tools to control cellular processes. We developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. A tetracycline aptamer was grafted onto the hammerhead ribozyme in such a way that ligand binding to the aptamers destroys a loop-loop interaction within the ribozyme thereby inhibiting ribozyme cleavage and allowing gene expression. The advantage of the presented regulatory system is its independence of any regulatory proteins. The stable integration of the ribozyme into the genome of HeLa cells indicates a low background activity in the absence of ligand. Furthermore, the ligand concentration required to robustly flip the switch does not affect cell viability and therefore allows a long-term application of the system. These properties turn the tetracycline-dependent ribozymes into a very promising tool for conditional gene expression in mammalian cells. PMID:25265236

  14. Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes.

    PubMed

    Beilstein, Kim; Wittmann, Alexander; Grez, Manuel; Suess, Beatrix

    2015-05-15

    Robust synthetic devices are requisite for the construction of synthetic genetic circuits and important scientific and technological tools to control cellular processes. We developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. A tetracycline aptamer was grafted onto the hammerhead ribozyme in such a way that ligand binding to the aptamers destroys a loop-loop interaction within the ribozyme thereby inhibiting ribozyme cleavage and allowing gene expression. The advantage of the presented regulatory system is its independence of any regulatory proteins. The stable integration of the ribozyme into the genome of HeLa cells indicates a low background activity in the absence of ligand. Furthermore, the ligand concentration required to robustly flip the switch does not affect cell viability and therefore allows a long-term application of the system. These properties turn the tetracycline-dependent ribozymes into a very promising tool for conditional gene expression in mammalian cells.

  15. Two group I ribozymes with different functions in a nuclear rDNA intron.

    PubMed Central

    Decatur, W A; Einvik, C; Johansen, S; Vogt, V M

    1995-01-01

    DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI. Images PMID:7556099

  16. Engineering of ribozyme-based riboswitches for mammalian cells.

    PubMed

    Wieland, Markus; Ausländer, David; Fussenegger, Martin

    2012-03-01

    Artificial RNA riboswitches--apart from protein-based gene regulation systems, which have been known about for a long time--have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer-ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells. PMID:22305857

  17. Installation of orthogonality to the interface that assembles two modular domains in the Tetrahymena group I ribozyme.

    PubMed

    Tanaka, Takahiro; Furuta, Hiroyuki; Ikawa, Yoshiya

    2014-04-01

    Two modular elements (P5abc and ΔP5) in the Tetrahymena group I ribozyme can be separated physically to generate a two-piece ribozyme derivative consisting of a separately prepared P5abc (P5 RNA) and the rest of the intron (ΔP5 RNA). Molecular recognition in the interface assembling P5 RNA and ΔP5 RNA is strong and specific, and the catalytic ability of the two-piece ribozyme is comparable to that of the parent unimolecular ribozyme. We designed alternative P14 (L5c-L2) interacting modules participating in the assembly of P5 and ΔP5 and investigated their ability in the context of complex formation of the two-piece ribozyme and in vivo splicing of the unimolecular intron ribozyme. Combined use of alternative P14 and L5b-P6 interacting modules provided robust orthogonality to the P5/ΔP5 assembly interface of the bimolecular complex.

  18. Recent developments in the hammerhead ribozyme field.

    PubMed Central

    Vaish, N K; Kore, A R; Eckstein, F

    1998-01-01

    Developments in the hammerhead ribozyme field during the last two years are reviewed here. New results on the specificity of this ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of ribozyme technology examples of the application of this ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction. PMID:9826743

  19. Group II introns: structure and catalytic versatility of large natural ribozymes.

    PubMed

    Lehmann, Karola; Schmidt, Udo

    2003-01-01

    Group II introns are large, natural catalytic RNAs or ribozymes that were discovered in organelles of certain protists, fungi, algae, and plants and more recently also in prokaryotic organisms. In vitro, some members were found to self-splice from their pre-RNAs by two consecutive transesterification reactions joining the flanking exons and releasing the intron in a typical lariat form. Apart from self-splicing, a variety of other in vitro activities have been detected for group II introns demonstrating their amazing catalytic versatility. Group II introns fold into a conserved secondary structure consisting of six domains radiating from a central wheel that brings the 5' and 3' splice junction into close proximity. Domain 1 is the largest domain that is assumed to deliver the molecular scaffold assembling the intron in its active structure, while domain 5 is the phylogenetically most conserved part that represents the active site of the ribozyme. In vivo, the splicing reaction of many, if not all group II introns is assisted by proteins either encoded by the introns themselves (maturases), or encoded by other genes of the host organisms. The host proteins known to date have additional cellular functions and seem to have been adapted for splicing during evolution. Some of the protein-encoding group II introns were also shown to act as mobile genetic elements. They can integrate efficiently into intronless alleles of the same gene (homing) and at much lower frequencies into ectopic sites (transposition). The mobility process depends on intron encoded protein functions (endonuclease and reverse transcriptase) and on the intron RNA. This review provides a comprehensive survey of the structure/function relationships and the reaction potential of group II introns, the structurally most complicated, but also most fascinating ribozymes when looking at their catalytic repertoire in vitro and in vivo.

  20. Protein-responsive ribozyme switches in eukaryotic cells.

    PubMed

    Kennedy, Andrew B; Vowles, James V; d'Espaux, Leo; Smolke, Christina D

    2014-10-29

    Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers.

  1. Protein-responsive ribozyme switches in eukaryotic cells

    PubMed Central

    Kennedy, Andrew B.; Vowles, James V.; d'Espaux, Leo; Smolke, Christina D.

    2014-01-01

    Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers. PMID:25274734

  2. Cryoenzymology of the hammerhead ribozyme.

    PubMed Central

    Feig, A L; Ammons, G E; Uhlenbeck, O C

    1998-01-01

    The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state. PMID:9769099

  3. Specificity of hammerhead ribozyme cleavage.

    PubMed Central

    Hertel, K J; Herschlag, D; Uhlenbeck, O C

    1996-01-01

    To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

  4. How do metal ions direct ribozyme folding?

    NASA Astrophysics Data System (ADS)

    Denesyuk, Natalia A.; Thirumalai, D.

    2015-10-01

    Ribozymes, which carry out phosphoryl-transfer reactions, often require Mg2+ ions for catalytic activity. The correct folding of the active site and ribozyme tertiary structure is also regulated by metal ions in a manner that is not fully understood. Here we employ coarse-grained molecular simulations to show that individual structural elements of the group I ribozyme from the bacterium Azoarcus form spontaneously in the unfolded ribozyme even at very low Mg2+ concentrations, and are transiently stabilized by the coordination of Mg2+ ions to specific nucleotides. However, competition for scarce Mg2+ and topological constraints that arise from chain connectivity prevent the complete folding of the ribozyme. A much higher Mg2+ concentration is required for complete folding of the ribozyme and stabilization of the active site. When Mg2+ is replaced by Ca2+ the ribozyme folds, but the active site remains unstable. Our results suggest that group I ribozymes utilize the same interactions with specific metal ligands for both structural stability and chemical activity.

  5. Increased Ribozyme Activity in Crowded Solutions*

    PubMed Central

    Desai, Ravi; Kilburn, Duncan; Lee, Hui-Ting; Woodson, Sarah A.

    2014-01-01

    Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg2+ concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg2+ threshold at which activity was detected and increased total RNA cleavage at high Mg2+ concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates. PMID:24337582

  6. Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Zimmerly, Steven

    2014-06-01

    Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.

  7. Predicted group II intron lineages E and F comprise catalytically active ribozymes.

    PubMed

    Nagy, Vivien; Pirakitikulr, Nathan; Zhou, Katherine Ismei; Chillón, Isabel; Luo, Jerome; Pyle, Anna Marie

    2013-09-01

    Group II introns are self-splicing, retrotransposable ribozymes that contribute to gene expression and evolution in most organisms. The ongoing identification of new group II introns and recent bioinformatic analyses have suggested that there are novel lineages, which include the group IIE and IIF introns. Because the function and biochemical activity of group IIE and IIF introns have never been experimentally tested and because these introns appear to have features that distinguish them from other introns, we set out to determine if they were indeed self-splicing, catalytically active RNA molecules. To this end, we transcribed and studied a set of diverse group IIE and IIF introns, quantitatively characterizing their in vitro self-splicing reactivity, ionic requirements, and reaction products. In addition, we used mutational analysis to determine the relative role of the EBS-IBS 1 and 2 recognition elements during splicing by these introns. We show that group IIE and IIF introns are indeed distinct active intron families, with different reactivities and structures. We show that the group IIE introns self-splice exclusively through the hydrolytic pathway, while group IIF introns can also catalyze transesterifications. Intriguingly, we observe one group IIF intron that forms circular intron. Finally, despite an apparent EBS2-IBS2 duplex in the sequences of these introns, we find that this interaction plays no role during self-splicing in vitro. It is now clear that the group IIE and IIF introns are functional ribozymes, with distinctive properties that may be useful for biotechnological applications, and which may contribute to the biology of host organisms.

  8. Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme

    PubMed Central

    Gumbs, Orlando H.; Padgett, Richard A.; Dayie, Kwaku T.

    2006-01-01

    We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1–3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1–10 mM MgCl2). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K d of 650 ± 250 nM, a K m of ∼300 nM, and a K cat of 0.02 min−1 under single turnover conditions. Within the ∼160-kDa D123–D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a well-behaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies. PMID:16894219

  9. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    PubMed Central

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo. PMID:22798264

  10. Crystal Structure of a Self-Spliced Group ll Intron

    SciTech Connect

    Toor,N.; Keating, K.; Taylor, S.; Pyle, A.

    2008-01-01

    Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resolution. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two-metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.

  11. Crystal Structure of a Self-Spliced Group II Intron

    SciTech Connect

    Toor, Navtej; Keating, Kevin S.; Taylor, Sean D.; Pyle, Anna Marie

    2008-04-10

    Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resolution. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two-metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.

  12. Self-Incorporation of Coenzymes by Ribozymes

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

  13. High-Throughput Mutational Analysis of a Twister Ribozyme.

    PubMed

    Kobori, Shungo; Yokobayashi, Yohei

    2016-08-22

    Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations. PMID:27461281

  14. Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.

    PubMed Central

    Beigelman, L; Karpeisky, A; Matulic-Adamic, J; Haeberli, P; Sweedler, D; Usman, N

    1995-01-01

    Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule. PMID:7501467

  15. Mapping L1 Ligase ribozyme conformational switch

    PubMed Central

    Giambaşu, George M.; Lee, Tai-Sung; Scott, William G.; York, Darrin M.

    2012-01-01

    L1 Ligase (L1L)molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5’-to-3’ phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA World hypothesis. L1L crystal structure captures two distinct conformations that differ by a re-orientation of one of the stems by around 80 Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution, and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a 3-state/2-step process. The first step involves a large-amplitude swing that re-orients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network. PMID:22771572

  16. Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria.

    PubMed

    Massel, Karen; Silke, Jordan R; Bonen, Linda

    2016-05-01

    Trans-splicing of discontinuous introns in plant mitochondria requires the assembly of independently-transcribed precursor RNAs into splicing-competent structures, and they are expected to be excised as Y-branched molecules ("broken lariats") because these introns belong to the group II ribozyme family. We now demonstrate that this is just one of several trans-splicing pathways for wheat mitochondrial nad1 intron 4 and nad5 intron 2; they also use a hydrolytic pathway and the liberated 5'-half-intron linear molecules are unexpectedly abundant in the RNA population. We also observe a third productive splicing pathway for nad5 intron 2 that yields full-length excised introns in which the termini are joined in vivo and possess non-encoded nucleotides. In the case of trans-splicing nad1 intron 1, which has a weakly-structured and poorly-conserved core sequence, excision appears to be solely through a hydrolytic pathway. When wheat embryos are germinated in the cold rather than at room temperature, an increased complexity in trans-splicing products is seen for nad1 intron 4, suggesting that there can be environmental effects on the RNA folding of bipartite introns. Our observations provide insights into intron evolution and the complexity of RNA processing events in plant mitochondria.

  17. Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria.

    PubMed

    Massel, Karen; Silke, Jordan R; Bonen, Linda

    2016-05-01

    Trans-splicing of discontinuous introns in plant mitochondria requires the assembly of independently-transcribed precursor RNAs into splicing-competent structures, and they are expected to be excised as Y-branched molecules ("broken lariats") because these introns belong to the group II ribozyme family. We now demonstrate that this is just one of several trans-splicing pathways for wheat mitochondrial nad1 intron 4 and nad5 intron 2; they also use a hydrolytic pathway and the liberated 5'-half-intron linear molecules are unexpectedly abundant in the RNA population. We also observe a third productive splicing pathway for nad5 intron 2 that yields full-length excised introns in which the termini are joined in vivo and possess non-encoded nucleotides. In the case of trans-splicing nad1 intron 1, which has a weakly-structured and poorly-conserved core sequence, excision appears to be solely through a hydrolytic pathway. When wheat embryos are germinated in the cold rather than at room temperature, an increased complexity in trans-splicing products is seen for nad1 intron 4, suggesting that there can be environmental effects on the RNA folding of bipartite introns. Our observations provide insights into intron evolution and the complexity of RNA processing events in plant mitochondria. PMID:26970277

  18. Crystallographic Analysis of Small Ribozymes and Riboswitches

    PubMed Central

    Lippa, Geoffrey M.; Liberman, Joseph A.; Jenkins, Jermaine L.; Krucinska, Jolanta; Salim, Mohammad; Wedekind, Joseph E.

    2016-01-01

    Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ1 riboswitch, and variants of a larger class II preQ1 riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, “first choice” RNA crystal screen compiled from our prior successes with commercially available screens. PMID:22315069

  19. Crystal Structure of the VS ribozyme

    PubMed Central

    Suslov, Nikolai B.; DasGupta, Saurja; Huang, Hao; Fuller, James R.; Lilley, David M.J.; Rice, Phoebe A.; Piccirilli, Joseph A.

    2015-01-01

    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. PMID:26414446

  20. In Vivo Evolution of a Catalytic RNA Couples Trans-Splicing to Translation

    PubMed Central

    Olson, Karen E.; Dolan, Gregory F.; Müller, Ulrich F.

    2014-01-01

    How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme’s trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell. PMID:24466112

  1. Structural and biochemical characterization of DSL ribozyme.

    PubMed

    Horie, Souta; Ikawa, Yoshiya; Inoue, Tan

    2006-01-01

    We recently reported on the molecular design and synthesis of a new RNA ligase ribozyme (DSL), whose active site was selected from a sequence library consisting of 30 random nucleotides set on a defined 3D structure of a designed RNA scaffold. In this study, we report on the structural and biochemical analyses of DSL. Structural analysis indicates that the active site, which consists of the selected sequence, attaches to the folded scaffold as designed. To see whether DSL resembles known ribozymes, a biochemical assay was performed. Metal-dependent kinetic studies suggest that the ligase requires Mg2+ ions. The replacement of Mg2+ with Co(NH3)6(3+) prohibits the reaction, indicating that DSL requires innersphere coordination of Mg2+ for a ligation reaction. The results show that DSL has requirements similar to those of previously reported catalytic RNAs.

  2. Solvent Structure and Hammerhead Ribozyme Catalysis

    PubMed Central

    Martick, Monika; Lee, Tai-Sung; York, Darrin M.; Scott, William G.

    2008-01-01

    SUMMARY Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 Å structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn2+ is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2′-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach. PMID:18420140

  3. An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

    PubMed Central

    Klauser, Benedikt; Hartig, Jörg S.

    2013-01-01

    An important requirement for achieving many goals of synthetic biology is the availability of a large repertoire of reprogrammable genetic switches and appropriate transmitter molecules. In addition to engineering genetic switches, the interconnection of individual switches becomes increasingly important for the construction of more complex genetic networks. In particular, RNA-based switches of gene expression have become a powerful tool to post-transcriptionally program genetic circuits. RNAs used for regulatory purposes have the advantage to transmit, sense, process and execute information. We have recently used the hammerhead ribozyme to control translation initiation in a small molecule-dependent fashion. In addition, riboregulators have been constructed in which a small RNA acts as transmitter molecule to control translation of a target mRNA. In this study, we combine both concepts and redesign the hammerhead ribozyme to sense small trans-acting RNAs (taRNAs) as input molecules resulting in repression of translation initiation in Escherichia coli. Importantly, our ribozyme-based expression platform is compatible with previously reported artificial taRNAs, which were reported to act as inducers of gene expression. In addition, we provide several insights into key requirements of riboregulatory systems, including the influences of varying transcriptional induction of the taRNA and mRNA transcripts, 5′-processing of taRNAs, as well as altering the secondary structure of the taRNA. In conclusion, we introduce an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits. PMID:23585277

  4. Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

    PubMed Central

    Forconi, Marcello; Benz-Moy, Tara; Gleitsman, Kristin Rule; Ruben, Eliza; Metz, Clyde; Herschlag, Daniel

    2012-01-01

    Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure–function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes. PMID:22543863

  5. Low Selection Pressure Aids the Evolution of Cooperative Ribozyme Mutations in Cells*

    PubMed Central

    Amini, Zhaleh N.; Müller, Ulrich F.

    2013-01-01

    Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs. PMID:24089519

  6. Biochemical analysis of hatchet self-cleaving ribozymes

    PubMed Central

    Li, Sanshu; Lünse, Christina E.; Harris, Kimberly A.; Breaker, Ronald R.

    2015-01-01

    Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg2+ to promote RNA strand scission with a maximum rate constant of ∼4 min−1. As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2′-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer. PMID:26385510

  7. Understanding splicing regulation through RNA splicing maps.

    PubMed

    Witten, Joshua T; Ule, Jernej

    2011-03-01

    Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

  8. The meaning of a minuscule ribozyme

    PubMed Central

    Yarus, Michael

    2011-01-01

    The smallest ribozyme that carries out a complex group transfer is the sequence GUGGC-3′, acting to aminoacylate GCCU-3′ (and host a manifold of further reactions) in the presence of substrate PheAMP. Here, I describe the enzymatic rate, the characterization of about 20 aminoacyl-RNA and peptidyl-RNA products and the pathways of these GUGGC/GCCU reactions. Finally, the topic is evolution, and the potential implications of these data for the advent of translation itself. PMID:21930581

  9. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  10. Intermolecular domain docking in the hairpin ribozyme

    PubMed Central

    Sumita, Minako; White, Neil A.; Julien, Kristine R.; Hoogstraten, Charles G.

    2013-01-01

    The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH3)63+. RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a “double conformational capture” model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding. PMID:23324606

  11. Molecular Crowding Accelerates Ribozyme Docking and Catalysis

    PubMed Central

    2015-01-01

    All biological processes take place in highly crowded cellular environments. However, the effect that molecular crowding agents have on the folding and catalytic properties of RNA molecules remains largely unknown. Here, we have combined single-molecule fluorescence resonance energy transfer (smFRET) and bulk cleavage assays to determine the effect of a molecular crowding agents on the folding and catalysis of a model RNA enzyme, the hairpin ribozyme. Our single-molecule data reveal that PEG favors the formation of the docked (active) structure by increasing the docking rate constant with increasing PEG concentrations. Furthermore, Mg2+ ion-induced folding in the presence of PEG occurs at concentrations ∼7-fold lower than in the absence of PEG, near the physiological range (∼1 mM). Lastly, bulk cleavage assays in the presence of the crowding agent show that the ribozyme’s activity increases while the heterogeneity decreases. Our data is consistent with the idea that molecular crowding plays an important role in the stabilization of ribozyme active conformations in vivo. PMID:25399908

  12. A ribozyme that triphosphorylates RNA 5′-hydroxyl groups

    PubMed Central

    Moretti, Janina E.; Müller, Ulrich F.

    2014-01-01

    The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5′-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min−1 under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5′-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source. PMID:24452796

  13. Chemistry and Biology of Self-Cleaving Ribozymes.

    PubMed

    Jimenez, Randi M; Polanco, Julio A; Lupták, Andrej

    2015-11-01

    Self-cleaving ribozymes were discovered 30 years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure, with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be used as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered. PMID:26481500

  14. Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes.

    PubMed

    Nomura, Yoko; Zhou, Linlin; Miu, Anh; Yokobayashi, Yohei

    2013-12-20

    We engineered small molecule responsive allosteric ribozymes based on the genomic hepatitis delta virus (HDV) ribozyme by replacing the P4-L4 stem-loop with an RNA aptamer through a connector stem. When embedded in the 3' untranslated region of a reporter gene mRNA, these RNA devices enabled regulation of cis-gene expression by theophylline and guanine by up to 29.5-fold in mammalian cell culture. Furthermore, a NOR logic gate device was constructed by placing two engineered ribozymes in tandem, demonstrating the modularity of the RNA devices. The significant improvement in the regulatory dynamic range (ON/OFF ratio) of the RNA devices based on the HDV ribozyme should provide new opportunities for practical applications. PMID:23697539

  15. Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

    PubMed Central

    Jankowsky, E; Schwenzer, B

    1996-01-01

    Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

  16. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

    PubMed Central

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E.; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-01-01

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits. PMID:25916845

  17. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    PubMed

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  18. Crystallization and preliminary diffraction analysis of a group I ribozyme from bacteriophage Twort

    SciTech Connect

    Chase, Elaine; Golden, Barbara L.

    2005-01-01

    A group I self-splicing RNA has been synthesized and cocrystallized with a four-nucleotide product RNA. Iodination of the product RNA produces a heavy-atom derivative suitable for structure determination. Group I introns are catalytic RNAs that are capable of performing a variety of phosphotransesterification reactions including self-splicing and RNA cleavage. The reactions are efficient, accurate and dependent only on the presence of guanosine-nucleotide substrate and sufficient magnesium ion to stabilize the structure of the RNA. To understand how the group I intron active-site facilitates catalysis, crystals of a 242-nucleotide ribozyme bound to a four-nucleotide product RNA have been produced that diffract to 3.6 Å resolution. The space group of these crystals is I2{sub 1}2{sub 1}2{sub 1} and the unit-cell parameters are a = 94.6, b = 141.0, c = 210.9 Å. A single heavy-atom derivative has been synthesized by covalent modification of the product RNA with iodine.

  19. Kinetic framework for ligation by an efficient RNA ligase ribozyme.

    PubMed

    Bergman, N H; Johnston, W K; Bartel, D P

    2000-03-21

    The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches. PMID:10715133

  20. A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA

    PubMed Central

    Martick, Monika; Horan, Lucas H.; Noller, Harry F.; Scott, William G.

    2008-01-01

    Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR1. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes4–7. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads8–10, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the poly-adenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals. PMID:18615019

  1. Efficient Ligation of the Schistosoma Hammerhead Ribozyme

    PubMed Central

    Canny, Marella D.; Jucker, Fiona M.; Pardi, Arthur

    2011-01-01

    The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2–3 at physiological Mg2+ ion concentrations (0.1 –1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme. PMID:17319693

  2. Probabilistic simple splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Heng, Fong Wan; Sarmin, Nor Haniza; Turaev, Sherzod

    2014-06-01

    A splicing system, one of the early theoretical models for DNA computing was introduced by Head in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings of DNA molecules at the specific recognition sites and attaches the prefix of the first string to the suffix of the second string, and the prefix of the second string to the suffix of the first string, thus yielding the new strings. For a specific type of splicing systems, namely the simple splicing systems, the recognition sites are the same for both strings of DNA molecules. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions have been considered for splicing systems in order to increase their computational power. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic simple splicing systems are investigated. We prove that probabilistic simple splicing systems can also increase the computational power of the splicing languages generated.

  3. Inhibition of the hammerhead ribozyme by neomycin.

    PubMed Central

    Stage, T K; Hertel, K J; Uhlenbeck, O C

    1995-01-01

    A series of antibiotics was tested for stimulation or inhibition of the hammerhead ribozyme cleavage reaction. Neomycin was found to be a potent inhibitor of the reaction with a Kl of 13.5 microM. Two hammerheads with well-characterized kinetics were used to determine which steps in the reaction mechanism were inhibited by neomycin. The data suggest that neomycin interacts preferentially with the enzyme-substrate complex and that this interaction leads to a reduction in the cleavage rate by stabilizing the ground state of the complex and destabilizing the transition state of the cleavage step. A comparison of neomycin with other aminoglycosides and inhibitors of hammerhead cleavage implies that the ammonium ions of neomycin are important for the antibiotic-hammerhead interaction. PMID:7489494

  4. Folding pathways of the Tetrahymena ribozyme.

    PubMed

    Mitchell, David; Russell, Rick

    2014-06-12

    Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs ≈ 0.6 min(-1), while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min(-1)). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the "choice" is enforced by energy barriers that grow larger as folding progresses.

  5. Folding pathways of the Tetrahymena ribozyme

    PubMed Central

    Mitchell, David; Russell, Rick

    2014-01-01

    Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs ≈ 0.6 min–1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here, we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min–1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the ‘choice’ is enforced by energy barriers that grow larger as folding progresses. PMID:24747051

  6. Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice

    PubMed Central

    Kashani-Sabet, Mohammed; Liu, Yong; Fong, Sylvia; Desprez, Pierre-Yves; Liu, Shuqing; Tu, Guanghuan; Nosrati, Mehdi; Handumrongkul, Chakkrapong; Liggitt, Denny; Thor, Ann D.; Debs, Robert J.

    2002-01-01

    To date, functional genomic studies have been confined to either cell-based assays or germline mutations, using transgenic or knockout animals. However, these approaches are often unable either to recapitulate complex biologic phenotypes, such as tumor metastasis, or to identify the specific genes and functional pathways that produce serious diseases in adult animals. Although the transcription factor NF-κB transactivates many metastasis-related genes in cells, the precise genes and functional-pathways through which NF-κB regulates metastasis in tumor-bearing hosts are poorly understood. Here, we show that the systemic delivery of plasmid-based ribozymes targeting NF-κB in adult, tumor-bearing mice suppressed NF-κB expression in metastatic melanoma cells, as well as in normal cell types, and significantly reduced metastatic spread. Plasmid-based ribozymes suppressed target-gene expression with sequence specificity not achievable by using synthetic oligonucleotide-based approaches. NF-κB seemed to regulate tumor metastasis through invasion-related, rather than angiogenesis-, cell-cycle- or apoptosis-related pathways in tumor-bearing mice. Furthermore, ribozymes targeting either of the NF-κB-regulated genes, integrin β3 or PECAM-1 (a ligand-receptor pair linked to cell adhesion), reduced tumor metastasis at a level comparable to NF-κB. These studies demonstrate the utility of gene targeting by means of systemic, plasmid-based ribozymes to dissect out the functional genomics of complex biologic phenotypes, including tumor metastasis. PMID:11891271

  7. [The use of nucleolytic enzymes (ribonucleases, polynucleotide phosphorylases and endonuclease from Serratia marcescens) for producing initial blocks of synthetic endoribonucleases].

    PubMed

    Kliagina, V P; Sedel'nikova, E A; Smolianinova, O A; Soboleva, I A; Khabarova, M I; Zhenodarova, S M

    1992-01-01

    The simplest variant of synthetic substrate-ribozyme complex has been proposed. The schemes of potential ribozyme "subunits" synthesis have been worked out: R1--GCUUGAAACAAA; R2--AAAAACUGAUGAAAGC. The macroscale synthesis of dinucleoside monophosphate ApU, GpC, CpU catalyzed by immobilized ribonucleases of different specificity and preparation of oligoadenylates by hydrolysis of poly-A in the presence of endonuclease Serratia marcescens, as well the synthesis of conservative sequences of potential ribozyme such as ApUpG, CpUpG, GpApU, ApApApG and others have been described.

  8. The evolution of spliced leader trans-splicing in nematodes.

    PubMed

    Pettitt, Jonathan; Harrison, Neale; Stansfield, Ian; Connolly, Bernadette; Müller, Berndt

    2010-08-01

    Spliced leader trans-splicing occurs in many primitive eukaryotes including nematodes. Most of our knowledge of trans-splicing in nematodes stems from the model organism Caenorhabditis elegans and relatives, and from work with Ascaris. Our investigation of spliced leader trans-splicing in distantly related Dorylaimia nematodes indicates that spliced-leader trans-splicing arose before the nematode phylum and suggests that the spliced leader RNA gene complements in extant nematodes have evolved from a common ancestor with a diverse set of spliced leader RNA genes.

  9. Biochemical analysis of pistol self-cleaving ribozymes

    PubMed Central

    Harris, Kimberly A.; Lünse, Christina E.; Li, Sanshu; Brewer, Kenneth I.; Breaker, Ronald R.

    2015-01-01

    Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min−1 under physiological Mg2+ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2′-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA. PMID:26385507

  10. Modulation of Group I Ribozyme Activity by Cationic Porphyrins

    PubMed Central

    Matsumura, Shigeyoshi; Ito, Tatsunobu; Tanaka, Takahiro; Furuta, Hiroyuki; Ikawa, Yoshiya

    2015-01-01

    The effects of cationic porphyrins on the catalytic activities of four group I ribozymes were investigated. A cationic porphyrin possessing four pyridinium moieties (pPyP) inhibited two group IC3 ribozymes (Syn Rz and Azo Rz) and a group IC1 ribozyme (Tet Rz). In the case of a group IA2 ribozyme (Td Rz), however, pPyP served not only as an inhibitor but also as an activator, and the effects of pPyP were dependent on its concentration. To analyze the structural and electronic factors determining the effects of pPyP on group I ribozymes, three cationic porphyrins (pPyNCP, pPyF4P, and TMPyP) were also examined. As interactions between small organic molecules and nucleic acids are attractive and important issues in biochemistry and biotechnology, this study contributes to the development of porphyrin-based molecules that can modulate functions of structured RNA molecules. PMID:25811638

  11. In vitro evolution of a ribozyme that contains 5-bromouridine

    NASA Technical Reports Server (NTRS)

    Dai, X.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

    2000-01-01

    The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

  12. Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-01

    The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+. PMID:1909564

  13. Selection of novel Mg(2+)-dependent self-cleaving ribozymes.

    PubMed Central

    Williams, K P; Ciafré, S; Tocchini-Valentini, G P

    1995-01-01

    Four RNA motifs are known that catalyse site-specific cleavage in the presence of Mg2+ ions, all discovered in natural RNAs. In a single in vitro selection experiment we have isolated representatives of five novel classes of Mg(2+)-dependent ribozymes. Small versions of three of these showed that a very simple internal loop type of secondary structure is responsible for the activity. One of these was synthesized in a bimolecular form, and compared directly with the hammerhead ribozyme; for the new ribozyme, the cleavage step of the reaction is much faster than the spontaneous rate of phosphodiester bond cleavage, yet substantially slower than that for the hammerhead. The results suggest that many more Mg(2+)-dependent self-cleaving RNA sequences can be found. Images PMID:7556098

  14. In-ice evolution of RNA polymerase ribozyme activity

    PubMed Central

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2014-01-01

    Mechanisms of molecular self-replication have the potential to shed light upon the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA World’. However, existing polymerase ribozymes lack the capacity to synthesise RNAs approaching their own size. Here we report the in vitro evolution of such catalysts directly in the RNA-stabilising medium of water-ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesise RNA in ices at temperatures as low as −19°C. Combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication. PMID:24256864

  15. Origins of the temperature dependence of hammerhead ribozyme catalysis.

    PubMed Central

    Peracchi, A

    1999-01-01

    The difficulties in interpreting the temperature dependence of protein enzyme reactions are well recognized. Here, the hammerhead ribozyme cleavage was investigated under single-turnover conditions between 0 and 60 degrees C as a model for RNA-catalyzed reactions. Under the adopted conditions, the chemical step appears to be rate-limiting. However, the observed rate of cleavage is affected by pre-catalytic equilibria involving deprotonation of an essential group and binding of at least one low-affinity Mg2+ion. Thus, the apparent entropy and enthalpy of activation include contributions from the temperature dependence of these equilibria, precluding a simple physical interpretation of the observed activation parameters. Similar pre-catalytic equilibria likely contribute to the observed activation parameters for ribozyme reactions in general. The Arrhenius plot for the hammerhead reaction is substantially curved over the temperature range considered, which suggests the occurrence of a conformational change of the ribozyme ground state around physiological temperatures. PMID:10390528

  16. In-ice evolution of RNA polymerase ribozyme activity

    NASA Astrophysics Data System (ADS)

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2013-12-01

    Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA world’. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5‧ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

  17. In-ice evolution of RNA polymerase ribozyme activity.

    PubMed

    Attwater, James; Wochner, Aniela; Holliger, Philipp

    2013-12-01

    Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial 'RNA world'. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5' extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication. PMID:24256864

  18. Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

    PubMed Central

    Komatsu, Y; Koizumi, M; Sekiguchi, A; Ohtsuka, E

    1993-01-01

    The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site. Images PMID:8441626

  19. Kinetic partitioning mechanism of HDV ribozyme folding

    SciTech Connect

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-14

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  20. Kinetic partitioning mechanism of HDV ribozyme folding

    NASA Astrophysics Data System (ADS)

    Chen, Jiawen; Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2014-01-01

    RNA folding kinetics is directly tied to RNA biological functions. We introduce here a new approach for predicting the folding kinetics of RNA secondary structure with pseudoknots. This approach is based on our previous established helix-based method for predicting the folding kinetics of RNA secondary structure. In this approach, the transition rates for an elementary step: (1) formation, (2) disruption of a helix stem, and (3) helix formation with concomitant partial melting of an incompatible helix, are calculated with the free energy landscape. The folding kinetics of the Hepatitis delta virus (HDV) ribozyme and the mutated sequences are studied with this method. The folding pathways are identified by recursive searching the states with high net flux-in(out) population starting from the native state. The theory results are in good agreement with that of the experiments. The results indicate that the bi-phasic folding kinetics for the wt HDV sequence is ascribed to the kinetic partitioning mechanism: Part of the population will quickly fold to the native state along the fast pathway, while another part of the population will fold along the slow pathway, in which the population is trapped in a non-native state. Single mutation not only changes the folding rate but also the folding pathway.

  1. Automated design of hammerhead ribozymes and validation by targeting the PABPN1 gene transcript

    PubMed Central

    Kharma, Nawwaf; Varin, Luc; Abu-Baker, Aida; Ouellet, Jonathan; Najeh, Sabrine; Ehdaeivand, Mohammad-Reza; Belmonte, Gabriel; Ambri, Anas; Rouleau, Guy; Perreault, Jonathan

    2016-01-01

    We present a new publicly accessible web-service, RiboSoft, which implements a comprehensive hammerhead ribozyme design procedure. It accepts as input a target sequence (and some design parameters) then generates a set of ranked hammerhead ribozymes, which target the input sequence. This paper describes the implemented procedure, which takes into consideration multiple objectives leading to a multi-objective ranking of the computer-generated ribozymes. Many ribozymes were assayed and validated, including four ribozymes targeting the transcript of a disease-causing gene (a mutant version of PABPN1). These four ribozymes were successfully tested in vitro and in vivo, for their ability to cleave the targeted transcript. The wet-lab positive results of the test are presented here demonstrating the real-world potential of both hammerhead ribozymes and RiboSoft. RiboSoft is freely available at the website http://ribosoft.fungalgenomics.ca/ribosoft/. PMID:26527730

  2. Structural studies on an internal loop from a hairpin ribozyme

    SciTech Connect

    Cai, Z.; SantaLucia, J. Jr.; Tinoco, I. Jr.

    1994-12-01

    Ribozymes, RNA enzymes, catalyze site-specific RNA cleavage and ligation reactions. We are studying the three-dimensional structure of a hairpin ribozyme derived from the minus strand of tobacco ring spot virus satellite RNA ((-)sTRSV), which has been engineering to specifically cleave the HIV-1 RNA. The minimum structure for the catalytic reaction involves a 50-nucleotide ribozyme and a 14-nucleotide substrate. The proposed secondary structure of the ribozyme-substrate complex consists of four short helices separated by two internal loops. The relatively large size (64-nucleotide) of the ribozyme-substrate complex presents formidable problems in solving the structure using NMR. Therefore we are studying smaller structural subunits of the complex. We are determining the high resolution structure of the symmetric internal loop involving the cleavage site and the flanking helices. One strand of the internal loop was selectively {sup 13}C-labeled at C8 of each purine and C6 of each pyrimidine. By using {sup 13}C-edited two-dimensional NMR, the proton NOESY spectrum was greatly simplified. This allowed unambiguous sequential proton resonance assignments along each strand. Three-dimensional {sup 1}-{sup 13}C HMQC-NOESY was used to further facilitate resonance assignments. We are also enzymatically synthesizing the entire 50-nucleotide ribozyme and will combine it with the {sup 13}C-labeled substrate. Through comparison of the NOE connectivities of the labeled nucleotides from the internal loop alone with those from the entire complex, the differences between the two structures can be elucidated.

  3. Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

    PubMed

    Tanaka, T; Inui, O; Dohi, N; Okada, N; Okada, H; Kikuchi, Y

    2001-07-01

    Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

  4. In vitro splicing reactions in Drosophila Kc nuclear extracts.

    PubMed

    Rio, Donald C

    2014-08-01

    This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1-2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea-acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA, the spliced mRNA, and the intermediates generated by the first step of splicing. For inefficient reactions, a more sensitive detection method, such as RNase protection, primer extension, or RT-PCR (reverse transcription-polymerase chain reaction), may be required.

  5. Introduction to cotranscriptional RNA splicing.

    PubMed

    Merkhofer, Evan C; Hu, Peter; Johnson, Tracy L

    2014-01-01

    The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.

  6. Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae.

    PubMed

    Klauser, Benedikt; Rehm, Charlotte; Summerer, Daniel; Hartig, Jörg S

    2015-01-01

    Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches. PMID:25605392

  7. The organization and contribution of helicases to RNA splicing.

    PubMed

    De, Inessa; Schmitzová, Jana; Pena, Vladimir

    2016-01-01

    Splicing is an essential step of gene expression. It occurs in two consecutive chemical reactions catalyzed by a large protein-RNA complex named the spliceosome. Assembled on the pre-mRNA substrate from five small nuclear proteins, the spliceosome acts as a protein-controlled ribozyme to catalyze the two reactions and finally dissociates into its components, which are re-used for a new round of splicing. Upon following this cyclic pathway, the spliceosome undergoes numerous intermediate stages that differ in composition as well as in their internal RNA-RNA and RNA-protein contacts. The driving forces and control mechanisms of these remodeling processes are provided by specific molecular motors called RNA helicases. While eight spliceosomal helicases are present in all organisms, higher eukaryotes contain five additional ones potentially required to drive a more intricate splicing pathway and link it to an RNA metabolism of increasing complexity. Spliceosomal helicases exhibit a notable structural diversity in their accessory domains and overall architecture, in accordance with the diversity of their task-specific functions. This review summarizes structure-function knowledge about all spliceosomal helicases, including the latter five, which traditionally are treated separately from the conserved ones. The implications of the structural characteristics of helicases for their functions, as well as for their structural communication within the multi-subunits environment of the spliceosome, are pointed out. PMID:26874649

  8. Computational Mutagenesis Studies of Hammerhead Ribozyme Catalysis

    PubMed Central

    Lee, Tai-Sung; York, Darrin M.

    2010-01-01

    Computational studies of the mutational effects at the C3, G8, and G5 positions of the hammerhead ribozyme (HHR) are reported based on a series of twenty four 100-ns molecular dynamics simulations of the native and mutated HHR in the reactant state and in an activated precursor state (G8:2′OH deprotonated). Invoking the assumptions that G12 acts as the general base while the 2′OH of G8 acts as a general acid, the simulations are able to explain the origins of experimentally observed mutational effects, including several that are not easily inferred from the crystal structure. Simulations suggest that the Watson-Crick base-pairing between G8 and C3, the hydrogen bond network between C17 and G5, and the base stacking interactions between G8 and C1.1, collectively, are key to maintaining an active site structure conducive for catalytic activity. Mutation-induced disruption of any of these interactions will adversely affect activity. The simulation results predict that the C3U/G8D double mutant, where D is 2,6-diaminopurine, will have a rescue effect relative to the corresponding single mutations. Two general conclusions about the simulations emerge from this work. Firstly, mutation simulations may require 30 ns or more to suitably relax such that the mutational effects become apparent. Secondly, in some cases, it is necessary to look beyond the reactant state in order to interpret mutational effects in terms of catalytically active structure. The present simulation results lead to better understanding of the origin of experimental mutational effects, and provide insight into the key conserved features necessary to maintain the integrity of the active site architecture. PMID:20812715

  9. Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

    PubMed Central

    Truong, David M.; Sidote, David J.; Russell, Rick; Lambowitz, Alan M.

    2013-01-01

    Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA (“ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites (“retrohoming”). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg2+ concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg2+ transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg2+ concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg2+ concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg2+ binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg2+-dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg2+ concentrations, with implications for evolution and potential applications in gene targeting. PMID:24043808

  10. Evolution in vitro: analysis of a lineage of ribozymes

    NASA Technical Reports Server (NTRS)

    Lehman, N.; Joyce, G. F.

    1993-01-01

    Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

  11. Amplification of RNA by an RNA polymerase ribozyme.

    PubMed

    Horning, David P; Joyce, Gerald F

    2016-08-30

    In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins. PMID:27528667

  12. Unusual metal ion catalysis in an acyl-transferase ribozyme.

    PubMed

    Suga, H; Cowan, J A; Szostak, J W

    1998-07-14

    Most studies of the roles of catalytic metal ions in ribozymes have focused on inner-sphere coordination of the divalent metal ions to the substrate or ribozyme. However, divalent metal ions are strongly hydrated in water, and some proteinenzymes, such as Escherichia coli RNase H and exonuclease III, are known to use metal cofactors in their fully hydrated form [Duffy, T. H., and Nowak, T. (1985) Biochemistry 24, 1152-1160; Jou, R., and Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686]. It is therefore important to consider the possibility of outer-sphere coordination of catalytic metal ions in ribozymes. We have used an exchange-inert metal complex, cobalt hexaammine, to show that the catalytic metal ion in an acyl-transferase ribozyme acts through outer-sphere coordination. Our studies provide an example of a fully hydrated Mg2+ ion that plays an essential role in ribozyme catalysis. Kinetic studies of wild-type and mutant ribozymes suggest that a pair of tandem G:U wobble base pairs adjacent to the reactive center constitute the metal-binding site. This result is consistent with recent crystallographic studies [Cate, J. H., and Doudna, J. A. (1996) Structure 4, 1221-1229; Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685; Cate, J. H., Hanna, R. L., and Doudna, J. A. (1997) Nat. Struct. Biol. 4, 553-558] showing that tandem wobble base pairs are good binding sites for metal hexaammines. We propose a model in which the catalytic metal ion is bound in the major groove of the tandem wobble base pairs, is precisely positioned by the ribozyme within the active site, and stabilizes the developing oxyanion in the transition state. Our results may have significant implications for understanding the mechanism of protein synthesis [Noller, H. F., Hoffarth, V., and Zimniak, L. (1992) Science 256, 1416-1419].

  13. NMR analysis of tertiary interactions in HDV ribozymes.

    PubMed

    Tanaka, Y; Hori, T; Tagaya, M; Katahira, M; Nishikawa, F; Sakamoto, T; Kurihara, Y; Nishikawa, S; Uesugi, S

    2000-01-01

    Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.

  14. Ribozyme motif structure mapped using random recombination and selection

    PubMed Central

    WANG, QING S.; UNRAU, PETER J.

    2005-01-01

    Isolating the core functional elements of an RNA is normally performed during the characterization of a new RNA in order to simplify further biochemical analysis. The removal of extraneous sequence is challenging and can lead to biases that result from the incomplete sampling of deletion variants. An impartial solution to this problem is to construct a library containing a large number of deletion constructs and to select functional RNA isolates that are at least as efficient as their full-length progenitors. Here, we use nonhomologous recombination and selection to isolate the catalytic core of a pyrimidine nucleotide synthase ribozyme. A variable-length pool of ~108 recombinant molecules that included deletions, inversions, and translocations of a 271-nucleotide-long ribozyme isolate was constructed by digesting and randomly religating its DNA genome. In vitro selection for functional ribozymes was then performed in a size-dependent and a size-independent manner. The final pools had nearly equivalent catalytic rates even though their length distributions were completely different, indicating that a diverse range of deletion constructs were functionally active. Four short sequence islands, requiring as little as 81 nt of sequence, were found within all of the truncated ribozymes and could be folded into a secondary structure consisting of three helix–loops. Our findings suggest that nonhomologous recombination is a highly efficient way to isolate a ribozyme’s core motif and could prove to be a useful method for evolving new ribozyme functions from pre-existing sequences in a manner that may have played an important role early in evolution. PMID:15703441

  15. Two stages splicing system

    NASA Astrophysics Data System (ADS)

    Mudaber, Mohammad Hassan; Yusof, Yuhani

    2015-05-01

    The study of the biological process of deoxyribonucleic acid (DNA) splicing system in a translucent approach was investigated in 2012 by Yusof under the framework of formal language theory. In this work, the concepts of splicing system in two stages as well as splicing languages are mathematically and biologically discussed. Additionally, some theorems based on recognition site factor of initial strings at the existence of two initial strings and two rules are provided via Yusof-Goode (Y-G) approach. Besides, an example is also given in showing the biological meaning of the introduced concept.

  16. How did alternative splicing evolve?

    PubMed

    Ast, Gil

    2004-10-01

    Alternative splicing creates transcriptome diversification, possibly leading to speciation. A large fraction of the protein-coding genes of multicellular organisms are alternatively spliced, although no regulated splicing has been detected in unicellular eukaryotes such as yeasts. A comparative analysis of unicellular and multicellular eukaryotic 5' splice sites has revealed important differences - the plasticity of the 5' splice sites of multicellular eukaryotes means that these sites can be used in both constitutive and alternative splicing, and for the regulation of the inclusion/skipping ratio in alternative splicing. So, alternative splicing might have originated as a result of relaxation of the 5' splice site recognition in organisms that originally could support only constitutive splicing. PMID:15510168

  17. A Hairpin Ribozyme Inhibits Expression of Diverse Strains of Human Immunodeficiency Virus Type 1

    NASA Astrophysics Data System (ADS)

    Yu, Mang; Ojwang, Joshua; Yamada, Osamu; Hampel, Arnold; Rapapport, Jay; Looney, David; Wong-Staal, Flossie

    1993-07-01

    Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the β-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

  18. Mutation analysis of the base-pair connecting two functional modules in the DSL ribozyme.

    PubMed

    Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya

    2008-01-01

    The class DSL ribozyme is one of artificial RNA enzymes generated by module-based molecular design. In the structure of this ribozyme, two most important functional modules are connected by a U-A base-pair. We have examined the possible importance of this base-pair by site-directed mutation experiments using the DSL-1S ribozyme and its derivative possessing altered modular organization. The analysis indicated that the DSL-1S ribozyme preferred U-A pair at the positions whereas the derivative preferred A-U pair.

  19. In vitro selection of allosteric ribozymes that sense the bacterial second messenger c-di-GMP.

    PubMed

    Furukawa, Kazuhiro; Gu, Hongzhou; Breaker, Ronald R

    2014-01-01

    Recently, a number of study have shown the ligand-dependent allosteric ribozymes can be harnessed as biosensors, high-throughput screening, and agents for the control of gene expression in vivo, called artificial riboswitches. In this chapter, we describe how in vitro selection can be used to create an allosteric ribozyme that senses bacterial second messenger cyclic-di-GMP (c-di-GMP). A hammerhead ribozyme was joined to a natural c-di-GMP class I riboswitch aptamer via communication modules. Both c-di-GMP-activating and -inhibiting ribozyme can be obtained by this approach. PMID:24549622

  20. Isolation of new ribozymes from a large pool of random sequences

    NASA Technical Reports Server (NTRS)

    Bartel, David P.; Szostak, Jack W.

    1993-01-01

    An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the attack of a 3'-hydroxyl on an adjacent 5'-triphosphate - a reaction similar to that employed by the familiar protein enzymes that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction rates 7 million times faster than the uncatalyzed reaction rate.

  1. Chromatin and alternative splicing.

    PubMed

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  2. Freeze-thaw cycles as drivers of complex ribozyme assembly

    PubMed Central

    Mutschler, Hannes; Wochner, Aniela; Holliger, Philipp

    2015-01-01

    The emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life. However, how such replicase ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication is unclear. Here we show that RNA polymerase ribozymes can assemble from simple catalytic networks of RNA oligomers no longer than 30 nucleotides. The entropically disfavoured assembly reaction is driven by iterative freeze-thaw cycles even in the absence of external activation chemistry. The steep temperature and concentration gradients of such cycles result in an RNA chaperone effect that enhances the otherwise only partially realized catalytic potential of the RNA oligomer pool by an order of magnitude. Our work outlines how cyclic physicochemical processes could have driven an expansion of RNA compositional and phenotypic complexity from simple oligomer pools. PMID:25991529

  3. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  4. A Triplex Ribozyme Expression System Based on a Single Hairpin Ribozyme

    PubMed Central

    Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A.

    2008-01-01

    Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with trans-cleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies. PMID:18707243

  5. Monitoring Retroviral RNA Dimerization In Vivo via Hammerhead Ribozyme Cleavage

    PubMed Central

    Pal, Bijay K.; Scherer, Lisa; Zelby, Laurie; Bertrand, Edouard; Rossi, John J.

    1998-01-01

    We have used a strategy for colocalization of Psi (Ψ)-tethered ribozymes and targets to demonstrate that Ψ sequences are capable of specific interaction in the cytoplasm of both packaging and nonpackaging cells. These results indicate that current in vitro dimerization models may have in vivo counterparts. The methodology used may be applied to further genetic analyses on Ψ domain interactions in vivo. PMID:9733882

  6. Local Neutral Networks Help Maintain Inaccurately Replicating Ribozymes

    PubMed Central

    Szilágyi, András; Kun, Ádám; Szathmáry, Eörs

    2014-01-01

    The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world. PMID:25299454

  7. The Novel Chemical Mechanism of the Twister Ribozyme.

    PubMed

    Wilson, Timothy J; Liu, Yijin; Domnick, Christof; Kath-Schorr, Stephanie; Lilley, David M J

    2016-05-18

    We describe the multifactorial origins of catalysis by the twister ribozyme. We provide evidence that the adenine immediately 3' to the scissile phosphate (A1) acts as a general acid. Substitution of ring nitrogen atoms indicates that very unusually the N3 of A1 is the proton donor to the oxyanion leaving group. A1 is accommodated in a specific binding pocket that raises its pKa toward neutrality, juxtaposes its N3 with the O5' to be protonated, and helps create the in-line trajectory required for nucleophilic attack. A1 performs general acid catalysis while G33 acts as a general base. A 100-fold stereospecific phosphorothioate effect at the scissile phosphate is consistent with a significant stabilization of the transition state by the ribozyme, and functional group substitution at G33 indicates that its exocyclic N2 interacts directly with the scissile phosphate. A model of the ribozyme active site is proposed that accommodates these catalytic strategies.

  8. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  9. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    PubMed Central

    Anella, Fabrizio; Danelon, Christophe

    2014-01-01

    The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA) vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment. PMID:25513761

  10. Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs.

    PubMed

    Cervera, Amelia; De la Peña, Marcos

    2014-11-01

    Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes. PMID:25135949

  11. Eukaryotic Penelope-Like Retroelements Encode Hammerhead Ribozyme Motifs

    PubMed Central

    Cervera, Amelia; De la Peña, Marcos

    2014-01-01

    Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes. PMID:25135949

  12. The Hammerhead Ribozyme: Structure, Catalysis and Gene Regulation

    PubMed Central

    Scott, William G.; Horan, Lucas H.; Martick, Monika

    2014-01-01

    The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme’s active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2′-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3′-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation. PMID:24156940

  13. The Novel Chemical Mechanism of the Twister Ribozyme.

    PubMed

    Wilson, Timothy J; Liu, Yijin; Domnick, Christof; Kath-Schorr, Stephanie; Lilley, David M J

    2016-05-18

    We describe the multifactorial origins of catalysis by the twister ribozyme. We provide evidence that the adenine immediately 3' to the scissile phosphate (A1) acts as a general acid. Substitution of ring nitrogen atoms indicates that very unusually the N3 of A1 is the proton donor to the oxyanion leaving group. A1 is accommodated in a specific binding pocket that raises its pKa toward neutrality, juxtaposes its N3 with the O5' to be protonated, and helps create the in-line trajectory required for nucleophilic attack. A1 performs general acid catalysis while G33 acts as a general base. A 100-fold stereospecific phosphorothioate effect at the scissile phosphate is consistent with a significant stabilization of the transition state by the ribozyme, and functional group substitution at G33 indicates that its exocyclic N2 interacts directly with the scissile phosphate. A model of the ribozyme active site is proposed that accommodates these catalytic strategies. PMID:27153229

  14. Local neutral networks help maintain inaccurately replicating ribozymes.

    PubMed

    Szilágyi, András; Kun, Ádám; Szathmáry, Eörs

    2014-01-01

    The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.

  15. RNA splicing and genes

    SciTech Connect

    Sharp, P.A.

    1988-11-25

    The splicing of long transcripts RNA (copied from DNA in the cell nucleus) into smaller specific mRNA is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing 5' and 3' splice sites. This complex contains the evolutionary highly conserved small nuclear RNAs (snRNAs) Us, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRANs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

  16. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    PubMed

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-01-01

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism. PMID:27404720

  17. Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme

    PubMed Central

    Trang, Phong; Vu, Gia-Phong; Lu, Sangwei; Wu, Jianguo; Liu, Fenyong

    2012-01-01

    Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G83 -> U83 and G340 -> A340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application. PMID:23300569

  18. Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

    PubMed

    Kim, Kihoon; Umamoto, Sean; Trang, Phong; Hai, Rong; Liu, Fenyong

    2004-03-01

    A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

  19. Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection.

    PubMed Central

    Tang, J; Breaker, R R

    1997-01-01

    We have designed a self-cleaving ribozyme construct that is rendered inactive during preparative in vitro transcription by allosteric interactions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a ribozyme whose catalytic rate can be controlled by ATP. Upon purification by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of "allosteric delay" was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for the hammerhead. Using in vitro selection, this comprehensive RNA pool was screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the core of naturally occurring hammerhead dominate the population of selected RNAs, indicating that the natural consensus sequence of this ribozyme is optimal for catalytic function. PMID:9257650

  20. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

    PubMed

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske

    2016-07-12

    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

  1. New classes of self-cleaving ribozymes revealed by comparative genomics analysis

    PubMed Central

    Weinberg, Zasha; Kim, Peter B.; Chen, Tony H.; Li, Sanshu; Harris, Kimberly A.; Lünse, Christina E.; Breaker, Ronald R.

    2015-01-01

    Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evident by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein this synteny was exploited to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. Three new self-cleaving classes, named twister sister, pistol and hatchet, have been identified from this collection, suggesting that even more ribozymes remain hidden in modern cells. PMID:26167874

  2. Transition State Features in the Hepatitis Delta Virus (HDV) Ribozyme Reaction Revealed by Atomic Perturbations

    PubMed Central

    Koo, Selene C.; Lu, Jun; Li, Nan-Sheng; Leung, Edward; Das, Subha R.; Harris, Michael E.; Piccirilli, Joseph A.

    2016-01-01

    Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the HDV ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group. PMID:26125657

  3. A widespread self-cleaving ribozyme class is revealed by bioinformatics

    PubMed Central

    Roth, Adam; Weinberg, Zasha; Chen, Andy G. Y.; Kim, Peter B.; Ames, Tyler D.; Breaker, Ronald R.

    2013-01-01

    Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs. PMID:24240507

  4. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  5. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

  6. SpliceDisease database: linking RNA splicing and disease.

    PubMed

    Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua

    2012-01-01

    RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation, we give direct links of the entry to the respective position/region in the genome browser. The users can freely browse, search and download the data in SpliceDisease at http://cmbi.bjmu.edu.cn/sdisease.

  7. A novel minimum ribozyme with oxidoreduction activity

    SciTech Connect

    Yanagawa, Hiroshi; Ogawa, Yoko ); Ueno, Masako; Sasaki, Kazuo; Sato, Toshio )

    1990-11-01

    A nucleoside catalyzing the oxidoreduction of NADH and K{sub 3}Fe(CN){sub 6} was isolated from Torula yeast RNA and also obtained in 0.05{percent} yield by a series of steps: SDS-phenol extraction, nuclease P{sub 1} digestion, alkaline phosphatase digestion, anion exchange chromatography, and HPLC on an ODS column. Its chemical structure was clearly determined at 5-hydroxycytidine, from the results of FAB-MS and {sup 1}H and {sup 13}C NMR spectroscopies. The mass spectra, chromatographic behavior, UV spectra, and NMR spectra of this nucleoside from natural and synthetic sources were identical. This is the first report of an RNA catalyst having catalytic activity except for the cleavage and ligation of phosphodiester bonds of RNA. That an RNA has oxidoreduction activity indicates new possibilities for RNAs as living molecules. 5-hydroxycytidine may be a vestige of RNAs that formerly possessed metabolizing ability.

  8. Cobalt(III)Hexaammine-Dependent Photocrosslinks in the Hairpin Ribozyme

    PubMed Central

    Kraemer-Chant, Christina M.; Heckman, Joyce E.; Lambert, Dominic; Burke, John M.

    2014-01-01

    We have utilized the hairpin ribozyme, an RNA enzyme whose structure has been solved by high-resolution methods, to develop a new tool for mapping nucleobase-stacking interactions and potential metal-binding sites in RNA molecules. This tool involves the photoactivation of a specifically bound cobalt(III)hexaammine molecule at wavelengths corresponding to excitation of the metal ion complex only; no base excitation is involved. The photoexcitation initiates a process which strongly promotes the formation of a novel covalent bond or crosslink between one base (termed the “first base”), which is close in space to the excited cobalt(III)hexaammine complex, and another base upon which the first base is closely stacked. These crosslinked species can be isolated and sequenced; their activities can be analyzed to ensure that the crosslinked structures represent an active conformation of the molecule. We have shown that, as in electron transfer in DNA, several criteria must be met to result in the successful formation of these crosslinks. These include the appropriate oxidation potential of the first donor base, the stacking and close interaction of the two donor bases involved in the crosslink, and the binding of a specific cobalt(III)hexaammine molecule to the first donor base. Additionally, we have determined that this crosslinking is pH-sensitive, although the cause of this sensitivity remains unknown. This tool has proven useful in the past for the analysis of the hairpin ribozyme folded structure, and has been applied to identifying potential metal-binding sites on the hairpin and extended hammerhead ribozymes. PMID:24295878

  9. Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

    PubMed Central

    Kore, A R; Vaish, N K; Kutzke, U; Eckstein, F

    1998-01-01

    The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH. PMID:9722629

  10. Speciation of a group I intron into a lariat capping ribozyme

    PubMed Central

    Meyer, Mélanie; Nielsen, Henrik; Oliéric, Vincent; Roblin, Pierre; Johansen, Steinar D.; Westhof, Eric; Masquida, Benoît

    2014-01-01

    The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2′,5′ branching reaction, leaving the 3′ product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching. PMID:24821772

  11. Applicability of PM3 to transphosphorylation reaction path: Toward designing a minimal ribozyme

    NASA Technical Reports Server (NTRS)

    Manchester, John I.; Shibata, Masayuki; Setlik, Robert F.; Ornstein, Rick L.; Rein, Robert

    1993-01-01

    A growing body of evidence shows that RNA can catalyze many of the reactions necessary both for replication of genetic material and the possible transition into the modern protein-based world. However, contemporary ribozymes are too large to have self-assembled from a prebiotic oligonucleotide pool. Still, it is likely that the major features of the earliest ribozymes have been preserved as molecular fossils in the catalytic RNA of today. Therefore, the search for a minimal ribozyme has been aimed at finding the necessary structural features of a modern ribozyme (Beaudry and Joyce, 1990). Both a three-dimensional model and quantum chemical calculations are required to quantitatively determine the effects of structural features of the ribozyme on the reaction it catalyzes. Using this model, quantum chemical calculations must be performed to determine quantitatively the effects of structural features on catalysis. Previous studies of the reaction path have been conducted at the ab initio level, but these methods are limited to small models due to enormous computational requirements. Semiempirical methods have been applied to large systems in the past; however, the accuracy of these methods depends largely on a simple model of the ribozyme-catalyzed reaction, or hydrolysis of phosphoric acid. We find that the results are qualitatively similar to ab initio results using large basis sets. Therefore, PM3 is suitable for studying the reaction path of the ribozyme-catalyzed reaction.

  12. In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.

    PubMed

    Lau, Matthew W L; Ferré-D'Amaré, Adrian R

    2016-08-15

    Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3min(-1) that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review. PMID:27130889

  13. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    NASA Technical Reports Server (NTRS)

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  14. Rapid generation of splicing reporters with pSpliceExpress

    PubMed Central

    Kishore, Shivendra; Khanna, Amit; Stamm, Stefan

    2008-01-01

    Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo. PMID:18930792

  15. Inventing and improving ribozyme function: rational design versus iterative selection methods

    NASA Technical Reports Server (NTRS)

    Breaker, R. R.; Joyce, G. F.

    1994-01-01

    Two major strategies for generating novel biological catalysts exist. One relies on our knowledge of biopolymer structure and function to aid in the 'rational design' of new enzymes. The other, often called 'irrational design', aims to generate new catalysts, in the absence of detailed physicochemical knowledge, by using selection methods to search a library of molecules for functional variants. Both strategies have been applied, with considerable success, to the remodeling of existing ribozymes and the development of ribozymes with novel catalytic function. The two strategies are by no means mutually exclusive, and are best applied in a complementary fashion to obtain ribozymes with the desired catalytic properties.

  16. Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.

    PubMed Central

    Sigurdsson, S T; Tuschl, T; Eckstein, F

    1995-01-01

    Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure. PMID:7489517

  17. Our favourite alternative splice site.

    PubMed

    Lerivray, Hubert; Méreau, Agnès; Osborne, H Beverley

    2006-05-01

    Alternative splicing is a widespread mechanism in mammals that generates several mRNAs from one gene, thereby creating genetic diversity of the genome. Variant splice patterns are often specific to different stages of development or particular tissues, and alternative splicing defects are being more frequently detected in genetic diseases and cancers. The increasingly important role of alternative splicing in the function and the regulation of cellular process makes it critical to have an easy-to-use data repository for the biological and medical research communities. We have compared web resources that give access to information on alternatively spliced genes, and the FAST DB (Friendly Alternative Splicing and Transcripts DataBase) site came out as our favourite.

  18. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  19. The neurogenetics of alternative splicing

    PubMed Central

    Vuong, Celine K.; Black, Douglas L.; Zheng, Sika

    2016-01-01

    Alternative precursor-mRNA splicing is a key mechanism for regulating gene expression in mammals and is controlled by specialized RNA-binding proteins. The misregulation of splicing is implicated in multiple neurological disorders. We describe recent mouse genetic studies of alternative splicing that reveal its critical role in both neuronal development and the function of mature neurons. We discuss the challenges in understanding the extensive genetic programmes controlled by proteins that regulate splicing, both during development and in the adult brain. PMID:27094079

  20. Continuous In Vitro Evolution of a Ribozyme that Catalyzes Three Successive Nucleotidyl Addition Reactions

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Wright, Martin C.; Joyce, Gerald F.

    2002-01-01

    Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(exp 406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5' yieldig 3' or a 3' yielding 5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

  1. Experimental evolution of complexity: in vitro emergence of intermolecular ribozyme interactions.

    PubMed Central

    Hanczyc, M M; Dorit, R L

    1998-01-01

    In the course of evolving variants of the Tetrahymena thermophila Group I ribozyme for improved DNA cleavage in vitro, we witnessed the unexpected emergence of a derived molecular species, capable of acting as a partner for the ribozyme, but no longer autocatalytic. This new RNA species exhibits a deletion in the catalytic core and participates in a productive intermolecular interaction with an active ribozyme, thus insuring its survival in the population. These novel RNA molecules have evolved a precise catalytic interaction with the Group I ribozyme and depend for their survival on the continued presence of active catalysts. This interaction hints at the complexity that may inevitably arise even in simple evolving systems. PMID:9510329

  2. Rational optimization of the DSL ligase ribozyme with GNRA/receptor interacting modules.

    PubMed

    Ishikawa, Junya; Matsumura, Shigeyoshi; Jaeger, Luc; Inoue, Tan; Furuta, Hiroyuki; Ikawa, Yoshiya

    2009-10-15

    The DSL ribozyme is a class of artificial ligase ribozymes with a highly modular architecture, which catalyzes template-directed RNA ligation on a helical substrate module that can be either covalently connected (cis-DSL) or physically separated (trans-DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively. In this study, we have rationally designed and analyzed the catalytic and self-assembly properties of several trans-DSL ribozymes with different sets of natural and artificial GNRA-receptor clamps. Two variants newly designed in this study showed significantly enhanced catalytic properties with respect of the original trans-DSL construct. While this work allows dissection of the turnover and catalytic properties of the trans-DSL ribozyme, it also emphasizes the remarkable modularity of RNA tertiary structure for nano-construction of complex functions.

  3. A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

    PubMed Central

    Wang, Bingbing; Salavati, Reza; Heidmann, Stefan; Stuart, Kenneth

    2002-01-01

    Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing. PMID:11991648

  4. Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

    PubMed Central

    PENEDO, J. CARLOS; WILSON, TIMOTHY J.; JAYASENA, SUMEDHA D.; KHVOROVA, ANASTASIA; LILLEY, DAVID M.J.

    2004-01-01

    It has been shown that the activity of the hammerhead ribozyme at μM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the μM range of magnesium ion concentration. Disruption of the loop–loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions. PMID:15100442

  5. Chemical synthesis of oligoribonucleotides containing 2-aminopurine: substrates for the investigation of ribozyme function

    NASA Technical Reports Server (NTRS)

    Doudna, J. A.; Szostak, J. W.; Rich, A.; Usman, N.

    1990-01-01

    The chemical synthesis of a fully protected ribonucleoside phosphoramidite, containing 2-aminopurine as the base component, and its incorporation into short oligoribonucleotides as substrates for an engineered ribozyme from Tetrahymena is described.

  6. Methods for Characterization of Alternative RNA Splicing.

    PubMed

    Harvey, Samuel E; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

  7. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  8. Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme.

    PubMed

    Saito, Hirohide; Suga, Hiroaki

    2002-12-01

    Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl-tRNA synthetase-like ribozyme (ARS ribozyme), which was evolved in vitro. This ribozyme can charge phenylalanine on tRNA in cis, where it is covalently attached to the 5'-end of tRNA (i.e. a form of precursor tRNA), as well as in trans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal-dependent kinetics, as well as structural mappings using Tb3+ in competition with Mg2+ or Co(NH3)6(3+), identified two potential metal-binding sites which are embedded near the tRNA-binding site. The high affinity metal-binding site can be filled with either Mg2+ or Co(NH3)6(3+) and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid-binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal-binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg2+ can enhance ribozyme activity, but Co(NH3)6(3+) cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.

  9. Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

    DOE PAGESBeta

    Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; Briber, R. M.; Woodson, S. A.

    2014-12-05

    The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

  10. Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

    SciTech Connect

    Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; Briber, R. M.; Woodson, S. A.

    2014-12-05

    The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.

  11. An in vitro evolved glmS ribozyme has the wildtype fold but loses coenzyme dependence

    PubMed Central

    Lau, Matthew W. L.; Ferré-D’Amaré, Adrian R.

    2014-01-01

    Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, use of GlcN6P is unexpected because all other characterized self-cleaving ribozymes employ RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P, and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wildtype fold but employs divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wildtype and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme- or cation-dependent. These results indicate that a few mutations can confer novel biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist. PMID:24096303

  12. Robust Suppression of HIV Replication by Intracellularly Expressed Reverse Transcriptase Aptamers Is Independent of Ribozyme Processing

    PubMed Central

    Lange, Margaret J; Sharma, Tarun K; Whatley, Angela S; Landon, Linda A; Tempesta, Michael A; Johnson, Marc C; Burke, Donald H

    2012-01-01

    RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two “minimal core” hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV. To evaluate the importance of the minimal ribozymes, we replaced them with extended, tertiary-stabilized hammerhead ribozymes with enhanced self-cleavage activity, in addition to noncleaving ribozymes with active site mutations. Both the active and inactive versions of the extended hammerhead ribozymes increased inhibition of pseudotyped virus, indicating that processing is not necessary for bioactivity. Clonal stable cell lines expressing aptamers from these modified constructs strongly suppressed infectious virus, and were more effective than minimal ribozymes at high viral multiplicity of infection (MOI). Tertiary stabilization greatly increased aptamer accumulation in viral and subcellular compartments, again regardless of self-cleavage capability. We therefore propose that the increased accumulation is responsible for increased suppression, that the bioactive form of the aptamer is one of the uncleaved or partially cleaved transcripts, and that tertiary stabilization increases transcript stability by reducing exonuclease degradation. PMID:22948672

  13. Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

    PubMed Central

    Lee, Hui-Ting; Kilburn, Duncan; Behrouzi, Reza; Briber, Robert M.; Woodson, Sarah A.

    2015-01-01

    The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly. PMID:25541198

  14. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

    PubMed Central

    Berzal-Herranz, A; Joseph, S; Chowrira, B M; Butcher, S E; Burke, J M

    1993-01-01

    In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived. Images PMID:8508779

  15. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

    PubMed Central

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-01-01

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. PMID:26114473

  16. Ribozyme Catalysis of Phosphodiester Bond Isomerization: The Hammerhead RNA and Its Relatives

    NASA Astrophysics Data System (ADS)

    Scott, William G.

    The hammerhead ribozyme is a comparatively small, self-cleaving RNA that has served as a prototype for understanding ribozyme catalysis. It has been intensively investigated using a variety of biochemical and biophysical techniques, yet for a simple ribozyme, it continues to yield surprises. A new structure of a full-length hammerhead ribozyme now reconciles over a decade of experimental discord and has helped to formulate a unified understanding of how it and other phosphodi-ester isomerase ribozymes function as catalysts. This whole family of ribozymes appears to exploit the chemistry of acid-base catalysis in a manner reminiscent of protein enzymes, such as RNase A, that catalyze similar reactions. Specifically, the roles of general base and general acid are often filled by the nucleotide functional groups themselves, in contrast to the originally anticipated ancillary structural role that RNA was thought to play, wherein the RNA was believed to be a passive scaffold upon which catalytically indispensable divalent metal ions might bind.

  17. Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

    PubMed Central

    Boots, Jennifer L.; Canny, Marella D.; Azimi, Ehsan; Pardi, Arthur

    2008-01-01

    The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed. PMID:18755844

  18. Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

    PubMed Central

    Inoue, Atsushi; Takagi, Yasuomi; Taira, Kazunari

    2004-01-01

    Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst. PMID:15302920

  19. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    PubMed

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  20. Stabilization of ribozyme-like cis-noncoding rRNAs induces apoptotic and nonapoptotic death in lung cells.

    PubMed

    Gee, M; Gu, Y; Fields, J R; Shiao, Y-H

    2012-01-01

    Bidirectional non-protein-coding RNAs are ubiquitously transcribed from the genome. Convergent sense and antisense transcripts may regulate each other. Here, we examined the convergent cis-noncoding rRNAs (nc-rRNAs) in A5 and E9 lung cancer models. Sense nc-rRNAs extending from rDNA intergenic region to internal transcribed spacer of around 10 kb in length were identified. nc-rRNAs in sense direction exhibited in vitro characteristics of ribozymes, namely, degradation upon incubation with MgCl(2) and stabilization by complementary oligonucleotides. Detection of endogenous cleavage-ligation products carrying internal deletion of hundreds to thousands nucleotides by massively parallel sequencing confirmed the catalytic properties. Transfection of oligonucleotides pairing with antisense nc-rRNAs stabilized both target and complementary transcripts, perturbed rRNA biogenesis, and induced massive cell death via apoptotic and/or nonapoptotic mechanisms depending on cell type and treatment. Oligonucleotides targeting cellular sense transcripts are less responsive. Spontaneously detached cells, though rare, also showed accumulation of nc-rRNAs and perturbation of rRNA biogenesis. Direct participation of nc-rRNAs in apoptotic and nonapoptotic death was demonstrated by transfection of synthetic nc-rRNAs encompassing the rDNA promoter. In sum, convergent cis-nc-rRNAs follow a feed-forward mechanism to regulate each other and rRNA biogenesis. This opens an opportunity to disrupt rRNA biogenesis, commonly upregulated in cancers, via inhibition of ribozyme-like activities in nc-rRNAs. PMID:22419110

  1. Stabilization of ribozyme-like cis-noncoding rRNAs induces apoptotic and nonapoptotic death in lung cells

    PubMed Central

    Gee, M; Gu, Y; Fields, J R; Shiao, Y-H

    2012-01-01

    Bidirectional non-protein-coding RNAs are ubiquitously transcribed from the genome. Convergent sense and antisense transcripts may regulate each other. Here, we examined the convergent cis-noncoding rRNAs (nc-rRNAs) in A5 and E9 lung cancer models. Sense nc-rRNAs extending from rDNA intergenic region to internal transcribed spacer of around 10 kb in length were identified. nc-rRNAs in sense direction exhibited in vitro characteristics of ribozymes, namely, degradation upon incubation with MgCl2 and stabilization by complementary oligonucleotides. Detection of endogenous cleavage-ligation products carrying internal deletion of hundreds to thousands nucleotides by massively parallel sequencing confirmed the catalytic properties. Transfection of oligonucleotides pairing with antisense nc-rRNAs stabilized both target and complementary transcripts, perturbed rRNA biogenesis, and induced massive cell death via apoptotic and/or nonapoptotic mechanisms depending on cell type and treatment. Oligonucleotides targeting cellular sense transcripts are less responsive. Spontaneously detached cells, though rare, also showed accumulation of nc-rRNAs and perturbation of rRNA biogenesis. Direct participation of nc-rRNAs in apoptotic and nonapoptotic death was demonstrated by transfection of synthetic nc-rRNAs encompassing the rDNA promoter. In sum, convergent cis-nc-rRNAs follow a feed-forward mechanism to regulate each other and rRNA biogenesis. This opens an opportunity to disrupt rRNA biogenesis, commonly upregulated in cancers, via inhibition of ribozyme-like activities in nc-rRNAs. PMID:22419110

  2. Selection of the Most Potent Specific On/Off Adaptor-Hepatitis Delta Virus Ribozymes for Use in Gene Targeting

    PubMed Central

    Lévesque, Michel V.; Rouleau, Samuel G.; Perreault, Jean-Pierre

    2016-01-01

    The Hepatitis Delta Virus (HDV) ribozyme, which is well adapted to the environment of the human cell, is an excellent candidate for the future development of gene-inactivation systems. On top of this, a new generation of HDV ribozymes now exists that benefits from the addition of a specific on/off adaptor (specifically the SOFA-HDV ribozymes) which greatly increases both the ribozyme’s specificity and its cleavage activity. Unlike RNAi and hammerhead ribozymes, the designing of SOFA-HDV ribozymes to cleave, in trans, given RNA species has never been the object of a systematic optimization study, even with their recent use for the gene knockdown of various targets. This report aims at both improving and clarifying the design process of SOFA-HDV ribozymes. Both the ribozyme and the targeted RNA substrate were analyzed in order to provide new criteria that are useful in the selection of the most potent SOFA-HDV ribozymes. The crucial features present in both the ribozyme’s biosensor and blocker, as well as at the target site, were identified and characterized. Simple rules were derived and tested using hepatitis C virus NS5B RNA as a model target. Overall, this method should promote the use of the SOFA-HDV ribozymes in a plethora of applications in both functional genomics and gene therapy. PMID:21793786

  3. NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Boisbouvier, Jérôme; Legault, Pascale

    2011-01-01

    The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem–loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson–Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme. PMID:21266483

  4. Cleavage of an amide bond by a ribozyme

    NASA Technical Reports Server (NTRS)

    Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1995-01-01

    A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

  5. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  6. Therapeutic targeting of splicing in cancer.

    PubMed

    Lee, Stanley Chun-Wei; Abdel-Wahab, Omar

    2016-09-01

    Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer. PMID:27603132

  7. Synthetic biology with RNA motifs.

    PubMed

    Saito, Hirohide; Inoue, Tan

    2009-02-01

    Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs. PMID:18775792

  8. Splicing in the human brain.

    PubMed

    Zaghlool, Ammar; Ameur, Adam; Cavelier, Lucia; Feuk, Lars

    2014-01-01

    It has become increasingly clear over the past decade that RNA has important functions in human cells beyond its role as an intermediate translator of DNA to protein. It is now known that RNA plays highly specific roles in pathways involved in regulatory, structural, and catalytic functions. The complexity of RNA production and regulation has become evident with the advent of high-throughput methods to study the transcriptome. Deep sequencing has revealed an enormous diversity of RNA types and transcript isoforms in human cells. The transcriptome of the human brain is particularly interesting as it contains more expressed genes than other tissues and also displays an extreme diversity of transcript isoforms, indicating that highly complex regulatory pathways are present in the brain. Several of these regulatory proteins are now identified, including RNA-binding proteins that are neuron specific. RNA-binding proteins also play important roles in regulating the splicing process and the temporal and spatial isoform production. While significant progress has been made in understanding the human transcriptome, many questions still remain regarding the basic mechanisms of splicing and subcellular localization of RNA. A long-standing question is to what extent the splicing of pre-mRNA is cotranscriptional and posttranscriptional, respectively. Recent data, including studies of the human brain, indicate that splicing is primarily cotranscriptional in human cells. This chapter describes the current understanding of splicing and splicing regulation in the human brain and discusses the recent global sequence-based analyses of transcription and splicing. PMID:25172473

  9. Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

    PubMed Central

    Sripathi, Kamali N.; Tay, Wendy W.; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G.

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2′-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. PMID:24854621

  10. Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

    PubMed Central

    Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

    2002-01-01

    Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

  11. Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

    PubMed Central

    Sioud, M

    1997-01-01

    The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs. PMID:9016562

  12. Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

    PubMed Central

    Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

    1997-01-01

    Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

  13. Impact of DNA gyrase inhibition by antisense ribozymes on rec A in E. coli.

    PubMed

    Shilpakala, Sainath Rao; Raghunathan, Malathi

    2009-09-01

    The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluoroquinolones.

  14. An expanded collection and refined consensus model of glmS ribozymes

    PubMed Central

    McCown, Phillip J.; Roth, Adam; Breaker, Ronald R.

    2011-01-01

    Self-cleaving glmS ribozymes selectively bind glucosamine-6-phosphate (GlcN6P) and use this metabolite as a cofactor to promote self-cleavage by internal phosphoester transfer. Representatives of the glmS ribozyme class are found in Gram-positive bacteria where they reside in the 5′ untranslated regions (UTRs) of glmS messenger RNAs that code for the essential enzyme L-glutamine:D-fructose-6-phosphate aminotransferase. By using comparative sequence analyses, we have expanded the number of glmS ribozyme representatives from 160 to 463. All but two glmS ribozymes are present in glmS mRNAs and most exhibit striking uniformity in sequence and structure, which are features that make representatives attractive targets for antibacterial drug development. However, our discovery of rare variants broadens the consensus sequence and structure model. For example, in the Deinococcus-Thermus phylum, several structural variants exist that carry additional stems within the catalytic core and changes to the architecture of core-supporting substructures. These findings reveal that glmS ribozymes have a broader phylogenetic distribution than previously known and suggest that additional rare structural variants may remain to be discovered. PMID:21367971

  15. RNA folding and the origins of catalytic activity in the hairpin ribozyme.

    PubMed

    Wilson, Timothy J; Nahas, Michelle; Araki, Lisa; Harusawa, Shinya; Ha, Taekjip; Lilley, David M J

    2007-01-01

    The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKa=6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.

  16. An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

    PubMed Central

    Herschlag, D; Khosla, M; Tsuchihashi, Z; Karpel, R L

    1994-01-01

    We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions. Images PMID:8026476

  17. Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

    PubMed Central

    Heinicke, Laurie A.; Bevilacqua, Philip C.

    2012-01-01

    Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. We report that the HDV ribozyme sequence alone can activate PKR. To elucidate the RNA structural basis for this, we prepared a number of HDV variants, including those with shortened or lengthened P4 pairing regions, with the anticipation that lengthening the P4 extension would yield a more potent activator since it would offer more base pairs of dsRNA. Surprisingly, the variant with a shortened P4 was the most potent activator. Through native gel mobility and enzymatic structure mapping experiments we implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease. PMID:23105000

  18. New tools provide a second look at HDV ribozyme structure, dynamics and cleavage

    PubMed Central

    Kapral, Gary J.; Jain, Swati; Noeske, Jonas; Doudna, Jennifer A.; Richardson, David C.; Richardson, Jane S.

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions. PMID:25326328

  19. Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

    PubMed Central

    Prommana, Parichat; Uthaipibull, Chairat; Wongsombat, Chayaphat; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Knuepfer, Ellen; Holder, Anthony A.; Shaw, Philip J.

    2013-01-01

    Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection. PMID:24023691

  20. Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

    PubMed Central

    Bassi, G S; Murchie, A I; Walter, F; Clegg, R M; Lilley, D M

    1997-01-01

    The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a single magnesium ion with an apparent association constant of approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA. PMID:9405376

  1. Conformational heterogeneity and the determinants of tertiary stabilization in the hammerhead ribozyme from Dolichopoda cave crickets

    PubMed Central

    Roychowdhury-Saha, Manami; Roychowdhury, Sugata

    2011-01-01

    Repetitive DNA elements in Dolichopoda cave cricket genomes contain extended hammerhead ribozymes that are functional in adult crickets, but that exhibit very low self-cleavage activity in vitro relative to other extended hammerhead ribozymes. We find that the parental ribozyme tends to misfold into alternate secondary structures in vitro, complicating analysis of contributions by specific nucleotides to activity under biologically relevant magnesium concentrations. However, minor sequence alterations that stabilize the active secondary structure, without altering candidate tertiary interacting nucleotides, boosted observed rates more than 50-fold (4.4 ± 1.7 min−1) and doubled the cleavage extent (>60%) in submillimolar magnesium. Productive alterations included flipping two base pairs in stem I, lengthening stem I and opening stem III to generate a trans-cleaving ribozyme. Specific peripheral nucleotides involved in tertiary stabilization were then identified through kinetic analysis for a series of sequence variants and by correlating plateau cleavage values with band intensity in native gel electrophoresis. These results demonstrate that conformational heterogeneity governs self-cleavage by the wild-type Dolichopoda hammerhead ribozyme in vitro, and they suggest a strategy for improving activity and enhancing the suitability of HHRz for intracellular and biotechnology applications. PMID:21712651

  2. Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

    PubMed Central

    Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

    1999-01-01

    A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

  3. An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

    PubMed Central

    Wang, L; Ruffner, D E

    1997-01-01

    The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly. PMID:9336468

  4. The Effect of Cytidine on the Structure and Function of an RNA Ligase Ribozyme

    NASA Technical Reports Server (NTRS)

    Rogers, Jeff; Joyce, Gerald F.

    2001-01-01

    A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random- sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5' -phosphodiester linkage with a catalytic rate of 0.016/min. The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32/min. The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

  5. A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection.

    PubMed

    Welch, P J; Tritz, R; Yei, S; Leavitt, M; Yu, M; Barber, J

    1996-11-01

    Two effective ribozymes (CR2 and CR4) that target HCV RNA 5' UTR and capsid gene regions were generated. Ribozyme cleavage was demonstrated in vitro, which can be enhanced by facilitator RNA molecules. In tissue culture cells, these two ribozymes can inhibit the expression of a cotransfected reporter gene containing HCV RNA target sequences. Furthermore, transduction of human hepatoma cells, HepG2, with retroviral vectors carrying CR2 or CR4 ribozymes enabled the cells to resist the infection by retroviral particles containing HCV target sequences. These results represent the first positive step towards the application of hairpin ribozymes in gene therapy for the treatment of HCV infection. PMID:9044745

  6. Alternative splicing regulation and cell lineage differentiation.

    PubMed

    Liu, Huan; He, Ling; Tang, Liling

    2012-11-01

    The alternative splicing of precursor mRNA is an essential mechanism for protein diversity. It plays important biological roles, such as proliferation, differentiation and development of cells. Furthermore, alternative splicing participates in the pathogenesis of diseases, including cancer. Thus, in-depth understanding of splicing regulation is of great significance. Regulation of alternative splicing is an extraordinary complicated process in which several signal molecules are at work. Besides the cis-elements and trans-factors, several lines of evidences suggest that other molecules, structures or process also regulate splicing, such as RNA structures, transcription and transcription factors, chromatin and protein. Meanwhile, increasing body of evidence shows that alternative splicing correlated closely to stem cell lineage differentiation. It means that there is a fundamental role for splicing in controlling regulatory program required for cell lineage differentiation. This review systematically sums up the regulation of alternative splicing and summarizes the splicing events during cell lineage differentiation of stem cells.

  7. Targeting RNA splicing for disease therapy.

    PubMed

    Havens, Mallory A; Duelli, Dominik M; Hastings, Michelle L

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics.

  8. Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis

    PubMed Central

    Chi, Young-In; Martick, Monika; Lares, Monica; Kim, Rosalind; Scott, William G; Kim, Sung-Hou

    2008-01-01

    We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures. PMID:18834200

  9. RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World

    PubMed Central

    Martin, Lyssa L.; Unrau, Peter J.; Müller, Ulrich F.

    2015-01-01

    The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory. PMID:25610978

  10. The complete VS ribozyme in solution studied by small-angle X-ray scattering

    SciTech Connect

    Lipfert, Jan; Ouellet, Jonathan; Norman, David G.; Doniach, Sebastian; Lilley, David M.J.

    2008-10-06

    We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.

  11. Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme.

    PubMed

    Nakano, Shu-ichi; Kitagawa, Yuichi; Yamashita, Hirofumi; Miyoshi, Daisuke; Sugimoto, Naoki

    2015-08-17

    The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl2 concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg(2+) concentration required for substrate cleavage. An inverse correlation was found between the MgCl2 concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg(2+) -induced structural transitions. The results suggest that the solution environment has various effects on the Mg(2+) interactions involved in the catalysis and global folding of the ribozyme.

  12. The complete VS ribozyme in solution studied by small-angle X-ray scattering

    PubMed Central

    Lipfert, Jan; Ouellet, Jonathan; Norman, David G.; Doniach, Sebastian; Lilley, David M.J.

    2010-01-01

    SUMMARY We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of transesterification in which adenine and guanine nucleobases contributed by the interacting loops combine together in general acid-base catalysis. PMID:18786398

  13. The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

    PubMed Central

    Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

    2013-01-01

    Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications

  14. The mitochondrial genome of the prasinophyte Prasinoderma coloniale reveals two trans-spliced group I introns in the large subunit rRNA gene.

    PubMed

    Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

    2013-01-01

    Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications

  15. Characterization of a native hammerhead ribozyme derived from schistosomes

    PubMed Central

    OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

    2005-01-01

    A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

  16. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing.

  17. Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions

    PubMed Central

    Strulson, Christopher A.; Yennawar, Neela H.; Rambo, Robert P.; Bevilacqua, Philip C.

    2013-01-01

    In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus (HDV)-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that under physiological free Mg2+ concentrations (~0.1 to 0.5 mM Mg2+), both crowders and cosolutes stimulate the rate of self-cleavage, up to ~6-fold, but that in 10 mM Mg2+—conditions widely used for in vitro ribozyme studies—these same additives have virtually no effect on self-cleavage rate. We further observe a dependence of self-cleavage rate on crowder size, wherein rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases in both low and high Mg2+ concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering (SAXS) experiments reveal a structural basis for this stimulation in that higher molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg2+ that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg2+ and no crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions. PMID:24187989

  18. Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

    PubMed Central

    Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

    2016-01-01

    The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction. PMID:26398724

  19. A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

    PubMed Central

    Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

    1995-01-01

    A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

  20. Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

    PubMed Central

    Soukup, G A; DeRose, E C; Koizumi, M; Breaker, R R

    2001-01-01

    Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches. PMID:11345431

  1. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    PubMed

    Wong, Stanley; Mosabbir, Abdullah A; Truong, Kevin

    2015-01-01

    Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins. PMID:26317656

  2. Design and Analysis of Hammerhead Ribozyme Activity Against an Artificial Gene Target

    PubMed Central

    Carter, James; Nawtaisong, Pruksa; Balaraman, Velmurugan; Fraser, Malcolm J.

    2014-01-01

    In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like the dengue virus RNA genomes. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays. PMID:24318886

  3. Evolution of splicing regulatory networks in Drosophila

    PubMed Central

    McManus, C. Joel; Coolon, Joseph D.; Eipper-Mains, Jodi; Wittkopp, Patricia J.; Graveley, Brenton R.

    2014-01-01

    The proteome expanding effects of alternative pre-mRNA splicing have had a profound impact on eukaryotic evolution. The events that create this diversity can be placed into four major classes: exon skipping, intron retention, alternative 5′ splice sites, and alternative 3′ splice sites. Although the regulatory mechanisms and evolutionary pressures among alternative splicing classes clearly differ, how these differences affect the evolution of splicing regulation remains poorly characterized. We used RNA-seq to investigate splicing differences in D. simulans, D. sechellia, and three strains of D. melanogaster. Regulation of exon skipping and tandem alternative 3′ splice sites (NAGNAGs) were more divergent than other splicing classes. Splicing regulation was most divergent in frame-preserving events and events in noncoding regions. We further determined the contributions of cis- and trans-acting changes in splicing regulatory networks by comparing allele-specific splicing in F1 interspecific hybrids, because differences in allele-specific splicing reflect changes in cis-regulatory element activity. We find that species-specific differences in intron retention and alternative splice site usage are primarily attributable to changes in cis-regulatory elements (median ∼80% cis), whereas species-specific exon skipping differences are driven by both cis- and trans-regulatory divergence (median ∼50% cis). These results help define the mechanisms and constraints that influence splicing regulatory evolution and show that networks regulating the four major classes of alternative splicing diverge through different genetic mechanisms. We propose a model in which differences in regulatory network architecture among classes of alternative splicing affect the evolution of splicing regulation. PMID:24515119

  4. Exploring ribozyme conformational changes with X-ray crystallography

    PubMed Central

    Spitale, Robert C.; Wedekind, Joseph E.

    2009-01-01

    Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the “gold standard” for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed ‘fourth generation’ radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of ‘conventional’ RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes; and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems. PMID:19559088

  5. Protein Trans-Splicing of Multiple Atypical Split Inteins Engineered from Natural Inteins

    PubMed Central

    Lin, Ying; Li, Mengmeng; Song, Huiling; Xu, Lingling; Meng, Qing; Liu, Xiang-Qin

    2013-01-01

    Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11–12 aa) N-intein fragment and S11 split inteins having a very small (6 aa) C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85–100%) of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ∼1.7×10−4 s−1 to ∼3.8×10−4 s−1, which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation. PMID:23593141

  6. Some Characterizations in Splicing Systems

    NASA Astrophysics Data System (ADS)

    Sarmin, Nor Haniza; Yusof, Yuhani; Wan Heng, Fong

    2010-11-01

    The splitting and recombinant of deoxyribonucleic acid or DNA by specified enzymes using concepts in Formal Language Theory was first mathematically modeled by Head in 1987. This splicing system, S can be presented as a set of initial string I over an alphabet A that acts upon 5' or 3' overhangs of restriction enzymes and can be simply viewed as S = (A, I, B, C). In this paper, a great interest in presenting some relations on certain types of splicing system namely null-context, uniform, simple, semi-simple, semi-null and Sk based on differentiating their rules are given as proposition, corollaries and counterexamples.

  7. The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

    PubMed Central

    Przybilski, Rita; Hammann, Christian

    2007-01-01

    Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. PMID:17666711

  8. Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing

    PubMed Central

    Kisseleva, Natalia; Kraut, Stefanie; Jäschke, Andres; Schiemann, Olav

    2007-01-01

    In ribozyme catalysis, metal ions are generally known to make structural and∕or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper Kd of 0.6±0.2 μM. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be ∼6 Å, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions. PMID:19404418

  9. Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

    NASA Technical Reports Server (NTRS)

    Tsang, Joyce; Joyce, Gerald F.

    1996-01-01

    In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

  10. The glmS ribozyme cofactor is a general acid-base catalyst.

    PubMed

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  11. Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage.

    PubMed

    Ren, Aiming; Vušurović, Nikola; Gebetsberger, Jennifer; Gao, Pu; Juen, Michael; Kreutz, Christoph; Micura, Ronald; Patel, Dinshaw J

    2016-09-01

    The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2'-O of G for attack on the adjacent to-be-cleaved P-O5' bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2'-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry. PMID:27398999

  12. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

  13. General acid-base catalysis mediated by nucleobases in the hairpin ribozyme

    PubMed Central

    Kath-Schorr, Stephanie; Wilson, Timothy J.; Li, Nan-Sheng; Lu, Jun; Piccirilli, Joseph A.; Lilley, David M. J.

    2012-01-01

    The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data. PMID:22958171

  14. The glmS Ribozyme Cofactor is a General Acid-Base Catalyst

    PubMed Central

    Viladoms, Julia; Fedor, Martha J.

    2012-01-01

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

  15. Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum

    PubMed Central

    LIANG, YU; ZHOU, YUELAN; YIN, WEIGUO; LI, YINGJU; YANG, QIULIN; GAO, YUAN; ZHANG, YUKUAI; YANG, YAOFEI; PENG, LI; XIAO, JIANHUA

    2012-01-01

    Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum. PMID:22246067

  16. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  17. A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage.

    PubMed Central

    Sekella, Phillip T; Rueda, David; Walter, Nils G

    2002-01-01

    Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 microM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector. PMID:12403463

  18. Nucleotide synthetase ribozymes may have emerged first in the RNA world

    PubMed Central

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2007-01-01

    Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

  19. Boosting of activity enhancement of K(+)-responsive quadruplex hammerhead ribozyme.

    PubMed

    Yamaoki, Yudai; Mashima, Tsukasa; Nagata, Takashi; Katahira, Masato

    2015-04-01

    Two second-generation quadruplex hammerhead ribozymes, whose activity enhances in response to K(+)via quadruplex formation of embedded r(GGA)3GG, were developed. Different strategies were applied to suppress basal activity when K(+) is absent. As a result, the activity enhancement upon the addition of K(+) has reached as high as 21-fold. PMID:25727931

  20. Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture

    PubMed Central

    Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2012-01-01

    Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure–function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing–loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (kcat/KM) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing–loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research. PMID:22086962

  1. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.

    PubMed

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-09-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  2. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

    PubMed Central

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-01-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  3. Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

    PubMed Central

    Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

    2000-01-01

    The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere

  4. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  5. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  6. Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

    SciTech Connect

    Cizdziel, P.E.; De Mars, M.; Murphy, E.C. Jr.

    1988-04-01

    The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33/sup 0/C or lower than at 37 to 41/sup 0/C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41/sup 0/C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. The authors exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39/sup 0/C. However, after a short (about 30-min) lag following a shift to 33/sup 0/C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA.

  7. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

  8. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  9. The genetics of splicing in neuroblastoma

    PubMed Central

    Chen, Justin; Hackett, Christopher S.; Zhang, Shile; Song, Young K.; Bell, Robert J.A.; Molinaro, Annette M.; Quigley, David A.; Balmain, Allan; Song, Jun S.; Costello, Joseph F.; Gustafson, W. Clay; Dyke, Terry Van; Kwok, Pui-Yan; Khan, Javed; Weiss, William A.

    2015-01-01

    Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest. Here we describe splicing quantitative trait loci (sQTL) associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. PMID:25637275

  10. Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

    PubMed Central

    Webb, Chiu-Ho T.; Nguyen, Dang; Myszka, Marie; Lupták, Andrej

    2016-01-01

    Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity – a “pinch” that results in rate acceleration by almost three orders of magnitude. Kinetic analysis of ribozymes with systematically altered length and stability of the peripheral domain revealed that about one third of its free energy of formation is used to lower an activation energy barrier, likely related to a rate-limiting conformational change leading to the pre-catalytic state. These findings provide a quantitative view of enzyme regulation by peripheral domains and may shed light on the energetics of allosteric regulation. PMID:27302490

  11. A novel strategy for selection of allosteric ribozymes yields RiboReporter sensors for caffeine and aspartame.

    PubMed

    Ferguson, Alicia; Boomer, Ryan M; Kurz, Markus; Keene, Sara C; Diener, John L; Keefe, Anthony D; Wilson, Charles; Cload, Sharon T

    2004-01-01

    We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter trade mark sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids.

  12. Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities

    PubMed Central

    Burke, Donald H; Greathouse, S Travis

    2005-01-01

    Background Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. Results Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25°C and 0.5 mM at 37°C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active. Conclusion Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the

  13. Development of a Functionally Minimized Mutant of the R3C Ligase Ribozyme Offers Insight into the Plausibility of the RNA World Hypothesis

    PubMed Central

    Kurihara, Eri; Uchida, Sayuri; Umehara, Takuya; Tamura, Koji

    2014-01-01

    The R3C ligase ribozyme is an artificial ligase ribozyme produced by modification of the ribozyme that lacks cytidine. Here, we attempted to modify the original R3C ribozyme (73 nucleotides) by reducing the number of nucleotides while maintaining the maximum possible catalytic efficiency. By partially deleting both the “grip” (P4 + P5) and “hammer” (P3) stem-loops, we found the critical border to retain activity comparable to that of full-length R3C. The three-way junction structure was necessary to maintain enzymatic function and the stability of the “grip” (P4 + P5) stem had a large influence on the catalytic activity of R3C. The final minimized ribozyme we obtained comprised ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Our findings suggest that the autocatalytic function in ribozymes is indeed possible to obtain using sequence lengths achievable with prebiotic synthesis. PMID:25256424

  14. A simple template-dependent ligase ribozyme as the RNA replicase emerging first in the RNA world.

    PubMed

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  15. A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

    PubMed

    Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

    2008-10-01

    The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

  16. The glmS ribozyme: use of a small molecule coenzyme by a gene-regulatory RNA.

    PubMed

    Ferré-D'Amaré, Adrian R

    2010-11-01

    The glmS ribozyme is the first known example of a natural ribozyme that has evolved to require binding of an exogenous small molecule for activity. In Gram-positive bacteria, this RNA domain is part of the messenger RNA (mRNA) encoding the essential enzyme that synthesizes glucosamine-6-phosphate (GlcN6P). When present at physiologic concentration, this small molecule binds to the glmS ribozyme and uncovers a latent self-cleavage activity that ultimately leads to degradation of the mRNA. Biochemical and structural studies reveal that the RNA adopts a rigid fold stabilized by three pseudoknots and the packing of a peripheral domain against the ribozyme core. GlcN6P binding to this pre-organized RNA does not induce conformational changes; rather, the small molecule functions as a coenzyme, providing a catalytically essential amine group to the active site. The ribozyme is not a passive player, however. Active site functional groups are essential for catalysis, even in the presence of GlcN6P. In addition to being a superb experimental system with which to analyze how RNA catalysts can exploit small molecule coenzymes to broaden their chemical versatility, the presence of the glmS ribozyme in numerous pathogenic bacteria make this RNA an attractive target for the development of new antibiotics and antibacterial strategies.

  17. Structural and Chemical Basis for Glucosamine 6-Phosphate Binding and Activation of the glmS Ribozyme

    SciTech Connect

    Cochrane, J.; Lipchock, S; Smith, K; Strobel, S

    2009-01-01

    The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation.

  18. Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

    PubMed Central

    Jankowsky, E; Strunk, G; Schwenzer, B

    1997-01-01

    Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

  19. Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

    PubMed Central

    de Silva, Chamaree; Walter, Nils G.

    2009-01-01

    Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer. PMID:19029309

  20. A Simple Template-Dependent Ligase Ribozyme as the RNA Replicase Emerging First in the RNA World

    NASA Astrophysics Data System (ADS)

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  1. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  2. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  3. Protein Trans-Splicing of an Atypical Split Intein Showing Structural Flexibility and Cross-Reactivity

    PubMed Central

    Song, Huiling; Meng, Qing; Liu, Xiang-Qin

    2012-01-01

    Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split intein, the intein fragments (IN and IC) can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small IN and a large IC is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large IC derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this IC could undergo trans-splicing in the presence of IN, and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this IC could trans-splice with small IN sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this IC could trans-splice even when the IN sequence was embedded in a nearly complete intein sequence, suggesting that the small IN could project out of the central pocket of the intein to become accessible to the IC. Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein. PMID:23024818

  4. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors. PMID:24336324

  5. Alternative Splicing in Plant Immunity

    PubMed Central

    Yang, Shengming; Tang, Fang; Zhu, Hongyan

    2014-01-01

    Alternative splicing (AS) occurs widely in plants and can provide the main source of transcriptome and proteome diversity in an organism. AS functions in a range of physiological processes, including plant disease resistance, but its biological roles and functional mechanisms remain poorly understood. Many plant disease resistance (R) genes undergo AS, and several R genes require alternatively spliced transcripts to produce R proteins that can specifically recognize pathogen invasion. In the finely-tuned process of R protein activation, the truncated isoforms generated by AS may participate in plant disease resistance either by suppressing the negative regulation of initiation of immunity, or by directly engaging in effector-triggered signaling. Although emerging research has shown the functional significance of AS in plant biotic stress responses, many aspects of this topic remain to be understood. Several interesting issues surrounding the AS of R genes, especially regarding its functional roles and regulation, will require innovative techniques and additional research to unravel. PMID:24918296

  6. Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption.

    PubMed

    Wimmer, K; Roca, X; Beiglböck, H; Callens, T; Etzler, J; Rao, A R; Krainer, A R; Fonatsch, C; Messiaen, L

    2007-06-01

    We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.

  7. Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

    PubMed Central

    Jiang, Shan; Xie, Qing; Zhang, Wei; Zhou, Xia-Qiu; Jin, You-Xin

    2005-01-01

    AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis. PMID:15996037

  8. Cryptic splice sites and split genes

    PubMed Central

    Kapustin, Yuri; Chan, Elcie; Sarkar, Rupa; Wong, Frederick; Vorechovsky, Igor; Winston, Robert M.; Tatusova, Tatiana; Dibb, Nick J.

    2011-01-01

    We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes. PMID:21470962

  9. RNA as an RNA Polymerase: Net Elongation of an RNA Primer Catalyzed by the Tetrahymena Ribozyme

    NASA Astrophysics Data System (ADS)

    Been, Michael D.; Cech, Thomas R.

    1988-03-01

    A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3', 5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.

  10. A recombinant RNA bacteriophage system to identify functionally important nucleotides in a self-cleaving ribozyme

    PubMed Central

    2014-01-01

    Background RNA bacteriophages like Qbeta and MS2 are well known for their high mutation rate, short infection cycle and strong selection against foreign inserts. The hammerhead ribozyme (HHRz) is a small self-cleaving RNA molecule whose active residues have previously been identified by mutational analysis of each individual base. Here the functionally important bases of HHRz were determined in a single screening experiment by inserting the HHRz into the genome of MS2. Findings The minimal HHRz of satellite Tobacco ringspot virus was cloned into the genome of RNA bacteriophage MS2. Sequence analysis of the surviving phages revealed that the majority had acquired single base-substitutions that apparently inactivated the HHRz. The positions of these substitutions exactly matched that of the previously determined core residues of the HHRz. Conclusions Natural selection against a ribozyme in the genome of MS2 can be used to quickly identify nucleotides required for self-cleavage. PMID:24946926

  11. Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner.

    PubMed

    Pan, Wei-Hua; Xin, Ping; Bui, Vuong; Clawson, Gary A

    2003-01-01

    A major limitation to the effectiveness of ribozymes is definition of accessible sites in targeted RNAs. Although library selection procedures have been developed, they are generally difficult to perform and have not been widely employed. Here we describe a selection technology that utilizes a randomized, active hammerhead ribozyme (Rz) library in an iterative manner. After two rounds of binding under inactive conditions, the selected, active Rz library is incubated with target RNA, and the sites of cleavage are identified on sequencing gels. We performed this library-selection protocol using human papillomavirus type 16 E6/E7 mRNA as target and constructed Rz targeted to the identified sites. Rz targeted to sites identified with this procedure were generally highly active in vitro and, more importantly, they were highly active in cell culture, whereas their catalytically inactive counterparts were not. This protocol can be used to identify a set of potential target sites within a relatively short time.

  12. Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution.

    PubMed

    Lie, Lively; Biliya, Shweta; Vannberg, Fredrik; Wartell, Roger M

    2016-03-01

    The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

  13. Thio Effects and an Unconventional Metal Ion Rescue in the Genomic HDV Ribozyme§

    PubMed Central

    Thaplyal, Pallavi; Ganguly, Abir; Golden, Barbara L.; Hammes-Schiffer, Sharon; Bevilacqua, Philip C.

    2013-01-01

    Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-RP oxygen at the scissile phosphate directly coordinates a catalytic Mg2+ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies on the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn2+. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd2+. Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg2+ or Cd2+, supporting that the pro-SP oxygen does not coordinate metal ions. The RP-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold effect and the slow-reacting phase displaying a thio effect of ~1,000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd2+ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd2+ inhibiting the oxo substrate but not the RP substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-RP oxygen, in line with experiments on the antigenomic HDV ribozyme. Experiments without divalent ions, with mutants that interfere with Mg2+ binding, or with C75 deleted suggest that the pro-RP oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-RP oxygen and the nucleophilic 2’- hydroxyl, supporting the experimental findings. PMID:24001219

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

    PubMed Central

    Bouchard, Patricia; Legault, Pascale

    2014-01-01

    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. PMID:25051972

  15. Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

    PubMed Central

    Roychowdhury-Saha, Manami; Burke, Donald H.

    2007-01-01

    Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role. PMID:17456566

  16. Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

    PubMed

    Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

    2015-05-15

    Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches. PMID:24871672

  17. Molecular aspects of DNA splicing system

    NASA Astrophysics Data System (ADS)

    Yusof, Yuhani; Lim, Wen Li; Goode, T. Elizabeth; Sarmin, Nor Haniza; Heng, Fong Wan; Wahab, Mohd Firdaus Abd

    2015-05-01

    The pioneer model of deoxyribonucleic acid (DNA) splicing system in a framework of Formal Language Theory was introduced by Head that led to the existence of other models of splicing system, namely Paun, Pixton and Yusof-Goode. These entire models are inspired by the molecular biological process of DNA splicing. Hence, this paper focuses on the translucent DNA splicing process, particularly on the generated language. Starting with some preliminaries in a limit graph, this paper also provides the experimental design with the predicted and actual result.

  18. The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

    PubMed

    Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

    2015-05-01

    When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

  19. Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline.

    PubMed

    Novak, Preston; Wang, Xiaobo; Ellenbecker, Mary; Feilzer, Sara; Voronina, Ekaterina

    2015-08-11

    Genetic interaction screens are an important approach for understanding complex regulatory networks governing development. We used a genetic interaction screen to identify cofactors of FBF-1 and FBF-2, RNA-binding proteins that regulate germline stem cell proliferation in Caenorhabditis elegans. We found that components of splicing machinery contribute to FBF activity as splicing factor knockdowns enhance sterility of fbf-1 and fbf-2 single mutants. This sterility phenocopied multiple aspects of loss of fbf function, suggesting that splicing factors contribute to stem cell maintenance. However, previous reports indicate that splicing factors instead promote the opposite cell fate, namely, differentiation. We explain this discrepancy by proposing that splicing factors facilitate overall RNA regulation in the germline. Indeed, we find that loss of splicing factors produces synthetic phenotypes with a mutation in another RNA regulator, FOG-1, but not with a mutation in a gene unrelated to posttranscriptional regulation (dhc-1). We conclude that inefficient pre-mRNA splicing may interfere with multiple posttranscriptional regulatory events, which has to be considered when interpreting results of genetic interaction screens.

  20. Synthetic oils

    NASA Technical Reports Server (NTRS)

    Hatton, R. E.

    1973-01-01

    Synthetic lubricants are discussed by chemical class and their general strengths and weaknesses in terms of lubrication properties are analyzed. Comparative ratings are given for 14 chemical classes and are used as a guide for lubricant selection. The effects of chemical structure on the properties of the lubricant are described with special emphasis on thermal stability. The diversity of synthetic lubricants which is provided by the wide range of properties permits many applications, some of which are reported.

  1. Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

    PubMed Central

    L'Huillier, P J; Soulier, S; Stinnakre, M G; Lepourry, L; Davis, S R; Mercier, J C; Vilotte, J L

    1996-01-01

    Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8692881

  2. A thiamin-utilizing ribozyme decarboxylates a pyruvate-like substrate

    NASA Astrophysics Data System (ADS)

    Cernak, Paul; Sen, Dipankar

    2013-11-01

    Vitamins are hypothesized to be relics of an RNA world, and were probably participants in RNA-mediated primordial metabolism. If catalytic RNAs, or ribozymes, could harness vitamin cofactors to aid their function in a manner similar to protein enzymes, it would enable them to catalyse a much larger set of chemical reactions. The cofactor thiamin diphosphate, a derivative of vitamin B1 (thiamin), is used by enzymes to catalyse difficult metabolic reactions, including decarboxylation of stable α-keto acids such as pyruvate. Here, we report a ribozyme that uses free thiamin to decarboxylate a pyruvate-based suicide substrate (LnkPB). Thiamin conjugated to biotin was used to isolate catalytic individuals from a pool of random-sequence RNAs attached to LnkPB. Analysis of a stable guanosine adduct obtained via digestion of an RNA sequence (clone dc4) showed the expected decarboxylation product. The discovery of a prototypic thiamin-utilizing ribozyme has implications for the role of RNA in orchestrating early metabolic cycles.

  3. Structural Investigation of the GlmS Ribozyme Bound to Its Catalytic Cofactor

    SciTech Connect

    Cochrane,J.; Lipchock, S.; Strobel, S.

    2007-01-01

    The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 {angstrom} resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.

  4. Properties of hepatitis delta virus ribozyme, which consists of three RNA oligomer strands.

    PubMed

    Sakamoto, T; Tanaka, Y; Kuwabara, T; Kim, M H; Kurihara, Y; Katahira, M; Uesugi, S

    1997-06-01

    Properties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg2+ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca2+ and Mn2+ ions were the most effective divalent cations and Mg2+ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40 degrees C. The Mg2+ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg2+ ion titration was monitored by CD. The CD data and the activity-Mg2+ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg2+ ions bind to one ribozyme unit.

  5. Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

    PubMed Central

    Alvarez-Salas, Luis M.; Cullinan, Amy E.; Siwkowski, Andrew; Hampel, Arnold; DiPaolo, Joseph A.

    1998-01-01

    HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization. PMID:9448307

  6. A Transition-State Interaction Shifts Nucleobase Ionization Toward Neutrality to Facilitate Small Ribozyme Catalysis

    PubMed Central

    Liberman, Joseph A.; Guo, Man; Jenkins, Jermaine L.; Krucinska, Jolanta; Chen, Yuanyuan; Carey, Paul R.; Wedekind, Joseph E.

    2012-01-01

    One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pKa values for the Ade38 N1-imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pKa value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pKa we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pKa values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pKa >0.8 log units relative to the precatalytic state. The agreement of the microscopic and macroscopic pKa values and the accompanying structural analysis support a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity. PMID:22989273

  7. Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

    PubMed Central

    Mustoe, Anthony M.; Al-Hashimi, Hashim M.; Brooks, Charles L.

    2016-01-01

    A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding. PMID:26481360

  8. A thiamin-utilizing ribozyme decarboxylates a pyruvate-like substrate.

    PubMed

    Cernak, Paul; Sen, Dipankar

    2013-11-01

    Vitamins are hypothesized to be relics of an RNA world, and were probably participants in RNA-mediated primordial metabolism. If catalytic RNAs, or ribozymes, could harness vitamin cofactors to aid their function in a manner similar to protein enzymes, it would enable them to catalyse a much larger set of chemical reactions. The cofactor thiamin diphosphate, a derivative of vitamin B1 (thiamin), is used by enzymes to catalyse difficult metabolic reactions, including decarboxylation of stable α-keto acids such as pyruvate. Here, we report a ribozyme that uses free thiamin to decarboxylate a pyruvate-based suicide substrate (LnkPB). Thiamin conjugated to biotin was used to isolate catalytic individuals from a pool of random-sequence RNAs attached to LnkPB. Analysis of a stable guanosine adduct obtained via digestion of an RNA sequence (clone dc4) showed the expected decarboxylation product. The discovery of a prototypic thiamin-utilizing ribozyme has implications for the role of RNA in orchestrating early metabolic cycles.

  9. Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

    PubMed Central

    Hammann, Christian; Norman, David G.; Lilley, David M. J.

    2001-01-01

    We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range. PMID:11331743

  10. Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

    PubMed Central

    Nakano, Shu-ichi; Kitagawa, Yuichi; Miyoshi, Daisuke; Sugimoto, Naoki

    2014-01-01

    Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol), small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds. PMID:25161873

  11. Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme.

    PubMed

    Krasovska, Maryna V; Sefcikova, Jana; Réblová, Kamila; Schneider, Bohdan; Walter, Nils G; Sponer, Jirí

    2006-07-15

    The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.

  12. Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

    PubMed Central

    Kraut, Stefanie; Bebenroth, Dirk; Nierth, Alexander; Kobitski, Andrei Y.; Nienhaus, G. Ulrich; Jäschke, Andres

    2012-01-01

    Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function. PMID:21976731

  13. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  14. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. PMID:25720307

  15. Use of a coenzyme by the glmS ribozyme-riboswitch suggests primordial expansion of RNA chemistry by small molecules.

    PubMed

    Ferré-D'Amaré, Adrian R

    2011-10-27

    The glmS ribozyme-riboswitch is the first known example of a naturally occurring catalytic RNA that employs a small molecule as a coenzyme. Binding of glucosamine-6-phosphate (GlcN6P) activates self-cleavage of the bacterial ribozyme, which is part of the mRNA encoding the metabolic enzyme GlcN6P-synthetase. Cleavage leads to negative feedback regulation. GlcN6P binds in the active site of the ribozyme, where its amine could function as a general acid and electrostatic catalyst. The ribozyme is pre-folded but inactive in the absence of GlcN6P, demonstrating it has evolved strict dependence on the exogenous small molecule. The ribozyme showcases the ability of RNA to co-opt non-covalently bound small molecules to expand its chemical repertoire. Analogue studies demonstrate that some molecules other than GlcN6P, such as l-serine (but not d-serine), can function as weak activators. This suggests how coenzyme use by RNA world ribozymes may have led to evolution of proteins. Primordial cofactor-dependent ribozymes may have evolved to bind their cofactors covalently. If amino acids were used as cofactors, this could have driven the evolution of RNA aminoacylation. The ability to make covalently bound peptide coenzymes may have further increased the fitness of such primordial ribozymes, providing a selective pressure for the invention of translation. PMID:21930586

  16. Use of a coenzyme by the glmS ribozyme-riboswitch suggests primordial expansion of RNA chemistry by small molecules.

    PubMed

    Ferré-D'Amaré, Adrian R

    2011-10-27

    The glmS ribozyme-riboswitch is the first known example of a naturally occurring catalytic RNA that employs a small molecule as a coenzyme. Binding of glucosamine-6-phosphate (GlcN6P) activates self-cleavage of the bacterial ribozyme, which is part of the mRNA encoding the metabolic enzyme GlcN6P-synthetase. Cleavage leads to negative feedback regulation. GlcN6P binds in the active site of the ribozyme, where its amine could function as a general acid and electrostatic catalyst. The ribozyme is pre-folded but inactive in the absence of GlcN6P, demonstrating it has evolved strict dependence on the exogenous small molecule. The ribozyme showcases the ability of RNA to co-opt non-covalently bound small molecules to expand its chemical repertoire. Analogue studies demonstrate that some molecules other than GlcN6P, such as l-serine (but not d-serine), can function as weak activators. This suggests how coenzyme use by RNA world ribozymes may have led to evolution of proteins. Primordial cofactor-dependent ribozymes may have evolved to bind their cofactors covalently. If amino acids were used as cofactors, this could have driven the evolution of RNA aminoacylation. The ability to make covalently bound peptide coenzymes may have further increased the fitness of such primordial ribozymes, providing a selective pressure for the invention of translation.

  17. Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells.

    PubMed Central

    Beck, J; Nassal, M

    1995-01-01

    Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cis-element present twice on this RNA is the encapsidation signal epsilon, a stem-loop structure that is critical for pregenome packaging and reverse transcription. Epsilon is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective in vitro cleavage inside epsilon by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon co-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient epsilon cleavage by the intracellularly expressed ribozymes was observed in Mg(2+)-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in cis and model RNAs lacking all HBV sequences except epsilon exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factor(s), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts. Images PMID:8559651

  18. Structure, dynamics, and function of the hammerhead ribozyme in bulk water and at a clay mineral surface from replica exchange molecular dynamics.

    PubMed

    Swadling, Jacob B; Wright, David W; Suter, James L; Coveney, Peter V

    2015-03-01

    Compared with proteins, the relationship between structure, dynamics, and function of RNA enzymes (known as ribozymes) is far less well understood, despite the fact that ribozymes are found in many organisms and are often conceived as "molecular fossils" of the first self-replicating molecules to have arisen on Earth. To investigate how ribozymal function is governed by structure and dynamics, we study the full hammerhead ribozyme in bulk water and in an aqueous clay mineral environment by computer simulation using replica-exchange molecular dynamics. Through extensive sampling of the major conformational states of the hammerhead ribozyme, we are able to show that the hammerhead manifests a free-energy landscape reminiscent of that which is well known in proteins, exhibiting a "funnel" topology that guides the ribozyme into its globally most stable conformation. The active-site geometry is found to be closely correlated to the tertiary structure of the ribozyme, thereby reconciling conflicts between previously proposed mechanisms for the self-scission of the hammerhead. The conformational analysis also accounts for the differences reported experimentally in the catalytic activity of the hammerhead ribozyme, which is reduced when interacting with clay minerals as compared with bulk water.

  19. Synthetic environments

    NASA Astrophysics Data System (ADS)

    Lukes, George E.; Cain, Joel M.

    1996-02-01

    The Advanced Distributed Simulation (ADS) Synthetic Environments Program seeks to create robust virtual worlds from operational terrain and environmental data sources of sufficient fidelity and currency to interact with the real world. While some applications can be met by direct exploitation of standard digital terrain data, more demanding applications -- particularly those support operations 'close to the ground' -- are well-served by emerging capabilities for 'value-adding' by the user working with controlled imagery. For users to rigorously refine and exploit controlled imagery within functionally different workstations they must have a shared framework to allow interoperability within and between these environments in terms of passing image and object coordinates and other information using a variety of validated sensor models. The Synthetic Environments Program is now being expanded to address rapid construction of virtual worlds with research initiatives in digital mapping, softcopy workstations, and cartographic image understanding. The Synthetic Environments Program is also participating in a joint initiative for a sensor model applications programer's interface (API) to ensure that a common controlled imagery exploitation framework is available to all researchers, developers and users. This presentation provides an introduction to ADS and the associated requirements for synthetic environments to support synthetic theaters of war. It provides a technical rationale for exploring applications of image understanding technology to automated cartography in support of ADS and related programs benefitting from automated analysis of mapping, earth resources and reconnaissance imagery. And it provides an overview and status of the joint initiative for a sensor model API.

  20. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations. PMID:26300000

  1. Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

    PubMed Central

    Boukis, L A; Bruzik, J P

    2001-01-01

    The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction. PMID:11421358

  2. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  3. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  4. Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?

    PubMed Central

    Müller, Sabine; Appel, Bettina; Balke, Darko; Hieronymus, Robert; Nübel, Claudia

    2016-01-01

    Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the help of high-throughput bioinformatic approaches. Understanding the biological role of the catalytic RNA motifs widely distributed in diverse genetic contexts belongs to the big challenges of future RNA research. PMID:27408700

  5. Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

    PubMed

    Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

    2016-08-19

    The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. PMID:27166370

  6. Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.

    PubMed Central

    Smith, S M; Maldarelli, F; Jeang, K T

    1997-01-01

    Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells. PMID:9371637

  7. A novel RNA-binding protein from Triturus carnifex identified by RNA-ligand screening with the newt hammerhead ribozyme

    PubMed Central

    Denti, Michela A.; Alba, A. Emilio Martínez de; Sägesser, Rudolf; Tsagris, Mina; Tabler, Martin

    2000-01-01

    The newt hammerhead ribozyme is transcribed from Satellite 2 DNA, which consists of tandemly repeated units of 330 bp. However, different transcripts are synthesized in different tissues. In all somatic tissues and in testes, dimeric and multimeric RNA transcripts are generated which, to some extent, self-cleave into monomers at the hammerhead domain. In ovaries, primarily a distinct monomeric unit is formed by transcription, which retains an intact hammerhead self-cleavage site. The ovarian monomeric RNA associates to form a 12S complex with proteins that are poorly characterised so far. In this work we identified NORA, a protein that binds the ovarian form of the newt ribozyme. We show that the newt ribozyme binds to the Escherichia coli-expressed protein, as well as to a protein of identical size that is found exclusively in newt ovaries. Also NORA mRNA was detectable only in ovary, but in neither somatic tissues nor testes. The tissue-specific expression of NORA is analogous to the ovary-specific transcription of the newt ribozyme. Although NORA was identified by its ability to bind to the newt ribozyme in the presence of a vast excess of carrier RNA, it was able to interact with certain other RNA probes. This novel RNA-binding protein does not contain any motif characteristic for RNA-binding proteins or any other known protein domain, but it shares a striking similarity with a rat resiniferatoxin-binding protein. PMID:10666442

  8. Functional coupling of transcription and splicing.

    PubMed

    Montes, Marta; Becerra, Soraya; Sánchez-Álvarez, Miguel; Suñé, Carlos

    2012-06-15

    The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing is a key mechanism to increase the number and complexity of proteins encoded by the genome. Evidence gathered in recent years has established that transcription and splicing are physically and functionally coupled and that this coupling may be an essential aspect of the regulation of splicing and alternative splicing. Recent advances in our understanding of transcription and of splicing regulation have uncovered the multiple interactions between components from both types of machinery. These interactions help to explain the functional coupling of RNAPII transcription and pre-mRNA alternative splicing for efficient and regulated gene expression at the molecular level. Recent technological advances, in addition to novel cell and molecular biology approaches, have led to the development of new tools for addressing mechanistic questions to achieve an integrated and global understanding of the functional coupling of RNAPII transcription and pre-mRNA alternative splicing. Here, we review major milestones and insights into RNA polymerase II transcription and pre-mRNA alternative splicing as well as new concepts and challenges that have arisen from multiple genome-wide approaches and analyses at the single-cell resolution.

  9. Synthetic Jets

    NASA Technical Reports Server (NTRS)

    Milanovic, Ivana M.

    2003-01-01

    Current investigation of synthetic jets and synthetic jets in cross-flow examined the effects of orifice geometry and dimensions, momentum-flux ratio, cluster of orifices, pitch and yaw angles as well as streamwise development of the flow field. This comprehensive study provided much needed experimental information related to the various control strategies. The results of the current investigation on isolated and clustered synthetic jets with and without cross-flow will be further analyzed and documented in detail. Presentations at national conferences and publication of peer- reviewed journal articles are also expected. Projected publications will present both the mean and turbulent properties of the flow field, comparisons made with the data available in an open literature, as well as recommendations for the future work.

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

    PubMed Central

    Buskiewicz, Iwona A.; Burke, John M.

    2012-01-01

    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. PMID:22274955

  11. The Characterizations of Different Splicing Systems

    NASA Astrophysics Data System (ADS)

    Karimi, Fariba; Sarmin, Nor Haniza; Heng, Fong Wan

    The concept of splicing system was first introduced by Head in 1987 to model the biological process of DNA recombination mathematically. This model was made on the basis of formal language theory which is a branch of applied discrete mathematics and theoretical computer science. In fact, splicing system treats DNA molecule and the recombinant behavior by restriction enzymes and ligases in the form of words and splicing rules respectively. The notion of splicing systems was taken into account from different points of view by many mathematicians. Several modified definitions have been introduced by many researchers. In this paper, some properties of different kinds of splicing systems are presented and their relationships are investigated. Furthermore, these results are illustrated by some examples.

  12. The connection between splicing and cancer.

    PubMed

    Srebrow, Anabella; Kornblihtt, Alberto R

    2006-07-01

    Alternative splicing is a crucial mechanism for generating protein diversity. Different splice variants of a given protein can display different and even antagonistic biological functions. Therefore, appropriate control of their synthesis is required to assure the complex orchestration of cellular processes within multicellular organisms. Mutations in cis-acting splicing elements or changes in the activity of constitutive or alternative splicing could have a profound regulatory proteins that compromise the accuracy of either impact on human pathogenesis, in particular in tumor development and progression. Mutations in splicing elements, for example, have been found in genes such as LKB1, KIT, CDH17, KLF6 and BRCA1, and changes in trans-acting regulators can affect the expression of genes such as Ron, RAC1 and CD44.

  13. Synthetic Astrobiology

    NASA Technical Reports Server (NTRS)

    Rothschild, Lynn J.

    2015-01-01

    Synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - has the potential to transform fields from pharmaceuticals to fuels. Our lab has focused on the potential of synthetic biology to revolutionize all three major parts of astrobiology: Where do we come from? Where are we going? and Are we alone? For the first and third, synthetic biology is allowing us to answer whether the evolutionary narrative that has played out on planet earth is likely to have been unique or universal. For example, in our lab we are re-evolving the biosynthetic pathways of amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids and developing techniques for the recovery of metals from spent electronics on other planetary bodies. In the future synthetic biology will play an increasing role in human activities both on earth, in fields as diverse as human health and the industrial production of novel bio-composites. Beyond earth, we will rely increasingly on biologically-provided life support, as we have throughout our evolutionary history. In order to do this, the field will build on two of the great contributions of astrobiology: studies of the origin of life and life in extreme environments.

  14. Synthetic Astrobiology

    NASA Technical Reports Server (NTRS)

    Rothschild, Lynn J.

    2016-01-01

    Synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - has the potential to transform fields from pharmaceuticals to fuels. Our lab has focused on the potential of synthetic biology to revolutionize all three major parts of astrobiology: Where do we come from? Where are we going? and Are we alone? For the first and third, synthetic biology is allowing us to answer whether the evolutionary narrative that has played out on planet earth is likely to have been unique or universal. For example, in our lab we are re-evolving the biosynthetic pathways of amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids and developing techniques for the recovery of metals from spent electronics on other planetary bodies. And what about the limits for life? Can we create organisms that expand the envelope for life? In the future synthetic biology will play an increasing role in human activities both on earth, in fields as diverse as human health and the industrial production of novel bio-composites. Beyond earth, we will rely increasingly on biologically-provided life support, as we have throughout our evolutionary history. In order to do this, the field will build on two of the great contributions of astrobiology: studies of the origin of life and life in extreme environments.

  15. Thiosulfate-Hydrogen Peroxide Redox Oscillator as pH Driver for Ribozyme Activity in the RNA World

    NASA Astrophysics Data System (ADS)

    Ball, Rowena; Brindley, John

    2016-03-01

    The RNA world of more than 3.7 billion years ago may have drawn on thermal and pH oscillations set up by the oxidation of thiosulfate by hydrogen peroxide (the THP oscillator) as a power source to drive replication. Since this primordial RNA also must have developed enzyme functionalities, in this work we examine the responses of two simple ribozymes to a THP periodic drive, using experimental rate and thermochemical data in a dynamical model for the coupled, self-consistent evolution of all reactants and intermediates. The resulting time traces show that ribozyme performance can be enhanced under pH cycling, and that thermal cycling may have been necessary to achieve large performance gains. We discuss three important ways in which the dynamic hydrogen peroxide medium may have acted as an agent for development of the RNA world towards a cellular world: proton gradients, resolution of the ribozyme versus replication paradox, and vesicle formation.

  16. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  17. Thiosulfate-Hydrogen Peroxide Redox Oscillator as pH Driver for Ribozyme Activity in the RNA World.

    PubMed

    Ball, Rowena; Brindley, John

    2016-03-01

    The RNA world of more than 3.7 billion years ago may have drawn on thermal and pH oscillations set up by the oxidation of thiosulfate by hydrogen peroxide (the THP oscillator) as a power source to drive replication. Since this primordial RNA also must have developed enzyme functionalities, in this work we examine the responses of two simple ribozymes to a THP periodic drive, using experimental rate and thermochemical data in a dynamical model for the coupled, self-consistent evolution of all reactants and intermediates. The resulting time traces show that ribozyme performance can be enhanced under pH cycling, and that thermal cycling may have been necessary to achieve large performance gains. We discuss three important ways in which the dynamic hydrogen peroxide medium may have acted as an agent for development of the RNA world towards a cellular world: proton gradients, resolution of the ribozyme versus replication paradox, and vesicle formation.

  18. Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets

    PubMed Central

    Barberan-Soler, Sergio; Zahler, Alan M.

    2008-01-01

    Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (∼18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 – now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream

  19. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

    Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions. PMID:18369186

  20. An Interspecific Plant Hybrid Shows Novel Changes in Parental Splice Forms of Genes for Splicing Factors

    PubMed Central

    Scascitelli, Moira; Cognet, Marie; Adams, Keith L.

    2010-01-01

    Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization. PMID:20100939

  1. A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

    PubMed Central

    Tabler, M; Homann, M; Tzortzakaki, S; Sczakiel, G

    1994-01-01

    Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. Images PMID:7937118

  2. Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme

    SciTech Connect

    Russell,R.; Tijerina, P.; Chadee, A.; Bhaskaran, H.

    2007-01-01

    The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant (E{sup {Delta}P5abc}) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E{sup {Delta}P5abc} forms its earliest stable tertiary structure on the millisecond time scale, {approx}5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E{sup {Delta}P5abc} in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E{sup {Delta}P5abc} ribozyme is much slower ({approx}0.6 min{sup -1}), in a manner similar to that of the wild type. Also similar, only a small fraction of E{sup {Delta}P5abc} attains the native state on this time scale under standard conditions at 25 {sup o}C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E{sup {Delta}P5abc} refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.

  3. Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme

    PubMed Central

    Lévesque, Dominique; Reymond, Cédric; Perreault, Jean-Pierre

    2012-01-01

    The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction. PMID:22768274

  4. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  5. Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

    PubMed Central

    Zhang, L; Liu, W; Grabowski, P J

    1999-01-01

    In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are

  6. Aberrant splicing and drug resistance in AML.

    PubMed

    de Necochea-Campion, Rosalia; Shouse, Geoffrey P; Zhou, Qi; Mirshahidi, Saied; Chen, Chien-Shing

    2016-01-01

    The advent of next-generation sequencing technologies has unveiled a new window into the heterogeneity of acute myeloid leukemia (AML). In particular, recurrent mutations in spliceosome machinery and genome-wide aberrant splicing events have been recognized as a prominent component of this disease. This review will focus on how these factors influence drug resistance through altered splicing of tumor suppressor and oncogenes and dysregulation of the apoptotic signaling network. A better understanding of these factors in disease progression is necessary to design appropriate therapeutic strategies recognizing specific alternatively spliced or mutated oncogenic targets. PMID:27613060

  7. Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

    PubMed Central

    Hendry, Philip; McCall, Maxine J; Stewart, Tom S; Lockett, Trevor J

    2004-01-01

    Background Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. Results A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent. Conclusion Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum. PMID:15588292

  8. Communication Between RNA Folding Domains Revealed by Folding of Circularly Permuted Ribozymes

    SciTech Connect

    Lease,R.; Adilakshmi, T.; Heilman-Miller, S.; Woodson, S.

    2007-01-01

    To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T1 digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

  9. Identification of the Catalytic Mg2+ Ion in the HDV Ribozyme

    PubMed Central

    Chen, Ji; Ganguly, Abir; Miswan, Zulaika; Hammes-Schiffer, Sharon; Bevilacqua, Philip C.; Golden, Barbara L.

    2013-01-01

    The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25•U20 reverse wobble. In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2’-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2’-hydroxyl. To test the role of the active site Mg2+ ion, we replaced the G25•U20 reverse wobble with an isosteric A25•C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg2+ binding could be adversely affected by the mutation. Kinetic analysis and molecular dynamics of the A25•C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant are inverted relative to the profiles for wild-type ribozyme, suggesting that the A25•C20 double mutant has lost the active site metal ion. Overall, these studies support a model wherein the partially hydrated Mg2+ positioned at the G25•U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile. PMID:23311293

  10. Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

    PubMed Central

    Eickbush, Danna G.; Burke, William D.; Eickbush, Thomas H.

    2013-01-01

    R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed. PMID:24066021

  11. Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures.

    PubMed

    El-Murr, Nizar; Maurel, Marie-Christine; Rihova, Martina; Vergne, Jacques; Hervé, Guy; Kato, Mikio; Kawamura, Kunio

    2012-09-01

    The "RNA world" hypothesis proposes that--early in the evolution of life--RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg(2+), which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg(2+) on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na(+) or K(+)), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

  12. Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation.

    PubMed

    Lee, Tai-Sung; Silva López, Carlos; Giambasu, George M; Martick, Monika; Scott, William G; York, Darrin M

    2008-03-12

    Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2'OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2'OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.

  13. Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

    PubMed Central

    Conaty, J; Hendry, P; Lockett, T

    1999-01-01

    In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system. PMID:10325431

  14. Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

    PubMed Central

    Lee, Tai-Sung; López, Carlos Silva; Giambaºu, George M.; Martick, Monika; Scott, William G.; York, Darrin M.

    2008-01-01

    Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2′OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2′OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods. PMID:18271579

  15. Structure-function relationships in the hammerhead ribozyme probed by base rescue.

    PubMed Central

    Peracchi, A; Matulic-Adamic, J; Wang, S; Beigelman, L; Herschlag, D

    1998-01-01

    We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs

  16. Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

    PubMed Central

    Gao, Run-Ping; Brigstock, David R

    2009-01-01

    AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase. PMID:19673024

  17. Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

    NASA Astrophysics Data System (ADS)

    El-Murr, Nizar; Maurel, Marie-Christine; Rihova, Martina; Vergne, Jacques; Hervé, Guy; Kato, Mikio; Kawamura, Kunio

    2012-09-01

    The "RNA world" hypothesis proposes that—early in the evolution of life—RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg2+, which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg2+ on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na+ or K+), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

  18. Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme

    PubMed Central

    Bouchard, Patricia; Lacroix-Labonté, Julie; Desjardins, Geneviève; Lampron, Philipe; Lisi, Véronique; Lemieux, Sébastien; Major, François; Legault, Pascale

    2008-01-01

    Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual 31P and 15N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface. PMID:18314503

  19. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  20. Regular languages, regular grammars and automata in splicing systems

    NASA Astrophysics Data System (ADS)

    Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

    2013-04-01

    Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

  1. Identifying Kinetic Barriers to Mechanical Unfolding of the T. thermophila Ribozyme

    NASA Astrophysics Data System (ADS)

    Onoa, Bibiana; Dumont, Sophie; Liphardt, Jan; Smith, Steven B.; Tinoco, Ignacio; Bustamante, Carlos

    2003-03-01

    Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are ``brittle,'' breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

  2. Ligation activity of fragmented ribozymes in frozen solution: implications for the RNA world

    PubMed Central

    Vlassov, Alexander V.; Johnston, Brian H.; Landweber, Laura F.; Kazakov, Sergei A.

    2004-01-01

    A vexing difficulty of the RNA world hypothesis is how RNA molecules of significant complexity could ever have evolved given their susceptibility to degradation. One way degradation might have been reduced is through low temperature. Here we report that truncated and fragmented derivatives of the hairpin ribozyme can catalyze ligation of a wide variety of RNA molecules to a given sequence in frozen solution despite having little or no activity under standard solution conditions. These results suggest that complex RNAs could have evolved in freezing environments on the early earth and perhaps elsewhere. PMID:15161960

  3. Distinct splicing signatures affect converged pathways in myelodysplastic syndrome patients carrying mutations in different splicing regulators.

    PubMed

    Qiu, Jinsong; Zhou, Bing; Thol, Felicitas; Zhou, Yu; Chen, Liang; Shao, Changwei; DeBoever, Christopher; Hou, Jiayi; Li, Hairi; Chaturvedi, Anuhar; Ganser, Arnold; Bejar, Rafael; Zhang, Dong-Er; Fu, Xiang-Dong; Heuser, Michael

    2016-10-01

    Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS. PMID:27492256

  4. The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2015-01-01

    As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III–IV–V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II–III–VI three-way junction and its adjoining stems. In addition, we localize Mg2+-binding sites within this structure using Mn2+-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg2+ ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge–bulge interaction and Mg2+ ions are critical for the stable folding of the II–III–VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II–III–VI junction that does not contain the II–VI bulge–bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme. PMID:26124200

  5. Alternative Splicing in Alzheimer’s Disease

    PubMed Central

    Love, Julia E.; Hayden, Eric J.; Rohn, Troy T.

    2015-01-01

    Neurodegenerative diseases have a variety of different genes contributing to their underlying pathology. Unfortunately, for many of these diseases it is not clear how changes in gene expression affect pathology. Transcriptome analysis of neurodegenerative diseases using ribonucleic acid sequencing (RNA Seq) and real time quantitative polymerase chain reaction (RT-qPCR) provides for a platform to allow investigators to determine the contribution of various genes to the disease phenotype. In Alzheimer’s disease (AD) there are several candidate genes reported that may be associated with the underlying pathology and are, in addition, alternatively spliced. Thus, AD is an ideal disease to examine how alternative splicing may affect pathology. In this context, genes of particular interest to AD pathology include the amyloid precursor protein (APP), TAU, and apolipoprotein E (APOE). Here, we review the evidence of alternative splicing of these genes in normal and AD patients, and recent therapeutic approaches to control splicing. PMID:26942228

  6. Shedding UV light on alternative splicing.

    PubMed

    Marengo, Matthew S; Garcia-Blanco, Mariano A

    2009-05-15

    After DNA damage, cells modulate pre-messenger RNA (pre-mRNA) splicing to induce an anti- or proapoptotic response. In this issue, Muñoz et al. (2009) uncover a cotranscriptional mechanism for activating alternative pre-mRNA splicing after ultraviolet irradiation that depends unexpectedly on hyperphosphorylation of the RNA polymerase II C-terminal domain and decreased rates of transcription elongation.

  7. Translational control of intron splicing in eukaryotes.

    PubMed

    Jaillon, Olivier; Bouhouche, Khaled; Gout, Jean-François; Aury, Jean-Marc; Noel, Benjamin; Saudemont, Baptiste; Nowacki, Mariusz; Serrano, Vincent; Porcel, Betina M; Ségurens, Béatrice; Le Mouël, Anne; Lepère, Gersende; Schächter, Vincent; Bétermier, Mireille; Cohen, Jean; Wincker, Patrick; Sperling, Linda; Duret, Laurent; Meyer, Eric

    2008-01-17

    Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

  8. The Alternative Splicing Mutation Database: a hub for investigations of alternative splicing using mutational evidence

    PubMed Central

    Bechtel, Jason M; Rajesh, Preeti; Ilikchyan, Irina; Deng, Ying; Mishra, Pankaj K; Wang, Qi; Wu, Xiaochun; Afonin, Kirill A; Grose, William E; Wang, Ye; Khuder, Sadik; Fedorov, Alexei

    2008-01-01

    Background Some mutations in the internal regions of exons occur within splicing enhancers and silencers, influencing the pattern of alternative splicing in the corresponding genes. To understand how these sequence changes affect splicing, we created a database of these mutations. Findings The Alternative Splicing Mutation Database (ASMD) serves as a repository for all exonic mutations not associated with splicing junctions that measurably change the pattern of alternative splicing. In this initial published release (version 1.2), only human sequences are present, but the ASMD will grow to include other organisms, (see Availability and requirements section for the ASMD web address). This relational database allows users to investigate connections between mutations and features of the surrounding sequences, including flanking sequences, RNA secondary structures and strengths of splice junctions. Splicing effects of the mutations are quantified by the relative presence of alternative mRNA isoforms with and without a given mutation. This measure is further categorized by the accuracy of the experimental methods employed. The database currently contains 170 mutations in 66 exons, yet these numbers increase regularly. We developed an algorithm to derive a table of oligonucleotide Splicing Potential (SP) values from the ASMD dataset. We present the SP concept and tools in detail in our corresponding article. Conclusion The current data set demonstrates that mutations affecting splicing are located throughout exons and might be enriched within local RNA secondary structures. Exons from the ASMD have below average splicing junction strength scores, but the difference is small and is judged not to be significant. PMID:18611286

  9. In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme

    PubMed Central

    Ren, Aiming; Košutić, Marija; Rajashankar, Kanagalaghatta R.; Frener, Marina; Santner, Tobias; Westhof, Eric; Micura, Ronald; Patel, Dinshaw J.

    2015-01-01

    Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modeled 2′-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5′ bond. Both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently-reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site. PMID:25410397

  10. A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

    PubMed Central

    Ren, Aiming; Flür, Sara; Wunderlich, Christoph; Mairhofer, Elisabeth; Vušurović, Nikola; Seikowski, Jan; Breuker, Kathrin; Höbartner, Claudia; Patel, Dinshaw J.; Kreutz, Christoph; Micura, Ronald

    2015-01-01

    Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH3-U5 modified twister ribozyme. PMID:26473980

  11. A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

    PubMed

    Košutić, Marija; Neuner, Sandro; Ren, Aiming; Flür, Sara; Wunderlich, Christoph; Mairhofer, Elisabeth; Vušurović, Nikola; Seikowski, Jan; Breuker, Kathrin; Höbartner, Claudia; Patel, Dinshaw J; Kreutz, Christoph; Micura, Ronald

    2015-12-01

    Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

  12. Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

    PubMed Central

    Pierce, M L; Ruffner, D E

    1998-01-01

    Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

  13. Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

    PubMed Central

    Hermann, T; Auffinger, P; Scott, W G; Westhof, E

    1997-01-01

    In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA. PMID:9254698

  14. Synthetic Cathinones ("Bath Salts")

    MedlinePlus

    ... still unknown about how synthetic cathinones affect the human brain. Researchers do know that synthetic cathinones are chemically ... of the chemicals in synthetic cathinones affect the human brain. Synthetic cathinones can cause: nosebleeds paranoia increased sociability ...

  15. Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

    PubMed Central

    Ben Aissa, Manel; April, Marie-Claude; Bergeron, Lucien-Junior; Perreault, Jean-Pierre; Levesque, Georges

    2012-01-01

    Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD. PMID:22482079

  16. Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

    PubMed Central

    Eiler, Daniel; Wang, Jimin; Steitz, Thomas A.

    2014-01-01

    Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket. PMID:25157168

  17. Determination of HDV ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics

    PubMed Central

    Kellerman, Daniel L; Simmons, Kandice S; Pedraza, Mayra; Piccirilli, Joseph A; York, Darrin M; Harris, Michael E

    2015-01-01

    Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the HDV ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis, and to test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed basepairing interaction. Mutants A78U, A78G and A79G retain significant catalytic activity, but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site, and illustrate multiple-substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions. PMID:25937290

  18. Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis

    PubMed Central

    Gaffarogullari, Ece Cazibe; Krause, André; Balbo, Jessica; Herten, Dirk-Peter; Jäschke, Andres

    2013-01-01

    The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (KD) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse. PMID:24448206

  19. Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

    PubMed Central

    Hoehlig, Kai; Bethge, Lucas; Klussmann, Sven

    2015-01-01

    A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications. PMID:25679211

  20. Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

    PubMed Central

    Mikulecky, Peter J.; Feig, Andrew L.

    2004-01-01

    In proteins, empirical correlations have shown that changes in heat capacity (ΔCP) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔCP on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔCPs of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔCP of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔCP by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔCP. We weigh the strengths and limitations of this method for determining ΔCP values. Finally, we discuss the data in light of the physical origins of the ΔCPs for RNA folding and consider their impact on biological function. PMID:15282329

  1. Ubiquitous presence of the hammerhead ribozyme motif along the tree of life

    PubMed Central

    de la Peña, Marcos; García-Robles, Inmaculada

    2010-01-01

    Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements. PMID:20705646

  2. RNA Tertiary Interactions Mediate Native Collapse of a Bacterial Group I Ribozyme

    SciTech Connect

    Chauhan, Seema; Caliskan, Gokhan; Briber, Robert M.; Perez-Salas, Ursula; Rangan, Prashanth; Thirumalai, D.; Woodson, Sarah A.

    2010-07-13

    Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg{sup 2+} concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg{sup 2+}-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg{sup 2+} dependence of collapse is similar to the Mg{sup 2+} dependence of helix assembly measured by partial ribonuclease T{sub 1} digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.

  3. Synthetic Cannabinoids.

    PubMed

    Mills, Brooke; Yepes, Andres; Nugent, Kenneth

    2015-07-01

    Synthetic cannabinoids (SCBs), also known under the brand names of "Spice," "K2," "herbal incense," "Cloud 9," "Mojo" and many others, are becoming a large public health concern due not only to their increasing use but also to their unpredictable toxicity and abuse potential. There are many types of SCBs, each having a unique binding affinity for cannabinoid receptors. Although both Δ-tetrahydrocannabinol (THC) and SCBs stimulate the same receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), studies have shown that SCBs are associated with higher rates of toxicity and hospital admissions than is natural cannabis. This is likely due to SCBs being direct agonists of the cannabinoid receptors, whereas THC is a partial agonist. Furthermore, the different chemical structures of SCBs found in Spice or K2 may interact in unpredictable ways to elicit previously unknown, and the commercial products may have unknown contaminants. The largest group of users is men in their 20s who participate in polydrug use. The most common reported toxicities with SCB use based on studies using Texas Poison Control records are tachycardia, agitation and irritability, drowsiness, hallucinations, delusions, hypertension, nausea, confusion, dizziness, vertigo and chest pain. Acute kidney injury has also been strongly associated with SCB use. Treatment mostly involves symptom management and supportive care. More research is needed to identify which contaminants are typically found in synthetic marijuana and to understand the interactions between different SBCs to better predict adverse health outcomes.

  4. Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress

    PubMed Central

    Ruan, Kai; Zhu, Yi; Li, Chong; Brazill, Jennifer M.; Zhai, R. Grace

    2015-01-01

    Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuroprotective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. Here we show that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. Our results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection. PMID:26616331

  5. Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress.

    PubMed

    Ruan, Kai; Zhu, Yi; Li, Chong; Brazill, Jennifer M; Zhai, R Grace

    2015-11-30

    Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuroprotective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. Here we show that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. Our results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection.

  6. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  7. Splicing therapy for neuromuscular disease☆

    PubMed Central

    Douglas, Andrew G.L.; Wood, Matthew J.A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. PMID:23631896

  8. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

  9. Linking Splicing to Pol II Transcription Stabilizes Pre-mRNAs and Influences Splicing Patterns

    PubMed Central

    Hicks, Martin J; Yang, Chin-Rang; Kotlajich, Matthew V

    2006-01-01

    RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs. PMID:16640457

  10. SplicePlot: a utility for visualizing splicing quantitative trait loci

    PubMed Central

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B.

    2014-01-01

    Summary: RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Availability and implementation: Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available. Contact: wu.eric.g@gmail.com or smontgom@stanford.edu PMID:24363378

  11. Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI).

    PubMed

    Schafer, Sebastian; Miao, Kui; Benson, Craig C; Heinig, Matthias; Cook, Stuart A; Hubner, Norbert

    2015-01-01

    Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon-centric manner and compared across conditions.

  12. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.

    PubMed

    Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

    2015-01-01

    To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine.

  13. A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection.

    PubMed

    Hoffmann, Steve; Otto, Christian; Doose, Gero; Tanzer, Andrea; Langenberger, David; Christ, Sabina; Kunz, Manfred; Holdt, Lesca M; Teupser, Daniel; Hackermüller, Jörg; Stadler, Peter F

    2014-02-10

    Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (http://www.bioinf.uni-leipzig.de/Software/segemehl/).

  14. Vitamin D and alternative splicing of RNA.

    PubMed

    Zhou, Rui; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Xu, Jianzhong; Adams, John S; Hewison, Martin

    2015-04-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  15. Variation in alternative splicing across human tissues

    PubMed Central

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793

  16. The behavior of bonded doubler splices for composite sandwich panels

    NASA Technical Reports Server (NTRS)

    Zeller, T. A.; Weisahaar, T. A.

    1980-01-01

    The results of an investigation into the behavior of adhesively bonded doubler splices of two composite material sandwich panels are presented. The splices are studied from three approaches: analytical; numerical (finite elements); and experimental. Several parameters that characterize the splice are developed to determine their influence upon joint strength. These parameters are: doubler overlap length; core stiffness; laminate bending stiffness; the size of the gap between the spliced sandwich panels; and room and elevated temperatures. Similarities and contrasts between these splices and the physically similar single and double lap joints are discussed. The results of this investigation suggest several possible approaches to improving the strength of the sandwich splices.

  17. Synthetic Brainbows

    PubMed Central

    Wan, Y.; Otsuna, H.; Hansen, C.

    2014-01-01

    Brainbow is a genetic engineering technique that randomly colorizes cells. Biological samples processed with this technique and imaged with confocal microscopy have distinctive colors for individual cells. Complex cellular structures can then be easily visualized. However, the complexity of the Brainbow technique limits its applications. In practice, most confocal microscopy scans use different florescence staining with typically at most three distinct cellular structures. These structures are often packed and obscure each other in rendered images making analysis difficult. In this paper, we leverage a process known as GPU framebuffer feedback loops to synthesize Brainbow-like images. In addition, we incorporate ID shuffing and Monte-Carlo sampling into our technique, so that it can be applied to single-channel confocal microscopy data. The synthesized Brainbow images are presented to domain experts with positive feedback. A user survey demonstrates that our synthetic Brainbow technique improves visualizations of volume data with complex structures for biologists. PMID:25018576

  18. Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

    PubMed Central

    Zhou, Jing-Min; Zhou, De-Min; Takagi, Yasuomi; Kasai, Yasuhiro; Inoue, Atsushi; Baba, Tadashi; Taira, Kazunari

    2002-01-01

    The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses. PMID:12034824

  19. Anti-beta s-ribozyme reduces beta s mRNA levels in transgenic mice: potential application to the gene therapy of sickle cell anemia.

    PubMed

    Alami, R; Gilman, J G; Feng, Y Q; Marmorato, A; Rochlin, I; Suzuka, S M; Fabry, M E; Nagel, R L; Bouhassira, E E

    1999-04-01

    Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human beta s mRNA. In this report, we have tested in vivo an anti-beta s hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human beta s gene. We then measured expression of the beta s transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of beta s mRNA in spleen stress reticulocytes (from 60.5 +/- 4.1% to 52.9 +/- 4.2%) and in the percentage of beta s globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of beta s chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.

  20. A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage

    SciTech Connect

    Chen, Jui-Hui; Yajima, Rieko; Chadalavada, Durga M.; Chase, Elaine; Bevilacqua, Philip C.; Golden, Barbara L.

    2010-11-01

    The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2{prime},3{prime}-cyclic phosphate and 5{prime}-hydroxyl termini. The active site nucleotide C75 has a pK{sub a} shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg{sup 2+} ion that helps activate the 2{prime}-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg{sup 2+} ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg{sup 2+} ion is also found within the active site where it interacts with a newly resolved G {center_dot} U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-RP oxygen of the scissile phosphate and the 2{prime}-hydroxyl nucleophile are inner-sphere ligands to the active site Mg{sup 2+} ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

  1. Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

    PubMed

    de Longevialle, Andéol Falcon; Small, Ian D; Lurin, Claire

    2010-07-01

    Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

  2. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  3. Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.

    PubMed Central

    König, H; Ponta, H; Herrlich, P

    1998-01-01

    Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions. PMID:9582284

  4. IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome.

    PubMed

    Shibata, Akihide; Okuno, Tatsuya; Rahman, Mohammad Alinoor; Azuma, Yoshiteru; Takeda, Jun-Ichi; Masuda, Akio; Selcen, Duygu; Engel, Andrew G; Ohno, Kinji

    2016-07-01

    Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3. PMID:27009626

  5. SpliceJumper: a classification-based approach for calling splicing junctions from RNA-seq data

    PubMed Central

    2015-01-01

    Background Next-generation RNA sequencing technologies have been widely applied in transcriptome profiling. This facilitates further studies of gene structure and expression on the genome wide scale. It is an important step to align reads to the reference genome and call out splicing junctions for the following analysis, such as the analysis of alternative splicing and isoform construction. However, because of the existence of introns, when RNA-seq reads are aligned to the reference genome, reads can not be fully mapped at splicing sites. Thus, it is challenging to align reads and call out splicing junctions accurately. Results In this paper, we present a classification based approach for calling splicing junctions from RNA-seq data, which is implemented in the program SpliceJumper. SpliceJumper uses a machine learning approach which combines multiple features extracted from RNA-seq data. We compare SpliceJumper with two existing RNA-seq analysis approaches, TopHat2 and MapSplice2, on both simulated and real data. Our results show that SpliceJumper outperforms TopHat2 and MapSplice2 in accuracy. The program SpliceJumper can be downloaded at https://github.com/Reedwarbler/SpliceJumper. PMID:26678515

  6. The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge–helix–bulge motifs of joined tRNA halves

    PubMed Central

    Randau, Lennart; Calvin, Kate; Hall, Michelle; Yuan, Jing; Podar, Mircea; Li, Hong; Söll, Dieter

    2005-01-01

    Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5′ and 3′ tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge–helix–bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate. PMID:16330750

  7. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  8. Epigenetics in alternative pre-mRNA splicing

    PubMed Central

    Luco, Reini F.; Allo, Mariano; Schor, Ignacio E.; Kornblihtt, Alberto R.; Misteli, Tom

    2010-01-01

    Alternative splicing plays critical roles in differentiation, development and disease and is a major source for protein diversity in higher eukaryotes. Traditionally, analysis of alternative splicing regulation has focused on RNA sequence elements and their associated factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation not only determines what parts of the genome are expressed, but also how they are spliced. PMID:21215366

  9. Coupling of Fast and Slow Modes in the Reaction Pathway of the Minimal Hammerhead Ribozyme Cleavage

    PubMed Central

    Radhakrishnan, Ravi

    2007-01-01

    By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg2+) ions, proceeds via an in-line-attack by CYT 17 O2′ on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack. PMID:17545240

  10. Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions.

    PubMed

    Hayden, Eric J

    2016-08-15

    RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis. PMID:27215494

  11. Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme.

    PubMed

    Shi, Xuesong; Bisaria, Namita; Benz-Moy, Tara L; Bonilla, Steve; Pavlichin, Dmitri S; Herschlag, Daniel

    2014-05-01

    We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA's topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes.

  12. Roles of Long-Range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme

    PubMed Central

    2015-01-01

    We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA’s topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes. PMID:24738560

  13. Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.

    PubMed

    Perrotta, Anne T; Wadkins, Timothy S; Been, Michael D

    2006-07-01

    In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Brønsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Brønsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.

  14. Ribozyme-Mediated Targeting of IκBγ Inhibits Melanoma Invasion and Metastasis

    PubMed Central

    Torabian, Sima Z.; de Semir, David; Nosrati, Mehdi; Bagheri, Sepideh; Dar, Altaf A.; Fong, Sylvia; Liu, Yong; Federman, Scot; Simko, Jeff; Haqq, Chris; Debs, Robert J.; Kashani-Sabet, Mohammed

    2009-01-01

    IκBγ is one member of a family of proteins that can inhibit the nuclear localization of nuclear factor-κB. However, the other specific functions of IκBγ are still poorly understood, and its effects on tumor metastasis have not yet been characterized. We examined the consequences of targeting IκBγ in melanoma cells using a hammerhead ribozyme. We developed stable transformant B16-F10 melanoma cell lines that express a ribozyme that targets mouse IκBγ (IκBγ-144-Rz). Tail-vein injection of B16-F10 cells that stably express IκBγ-144-Rz into mice resulted in a significant reduction of the metastatic potential of these cells. IκBγ-144-Rz-expressing B16 cells were shown to have increased transcriptional activity of nuclear factor-κB. We then showed that IκBγ-144-Rz-expressing cells demonstrated both reduced invasion and increased apoptosis, suggesting the existence of pathways through which IκBγ promotes melanoma metastasis. Using gene expression profiling, we identified a differentially expressed gene set that is regulated by the stable suppression of IκBγ that may participate in mediating its anti-metastatic effects; we also confirmed the altered expression levels of several of these genes by quantitative real time polymerase chain reaction. Plasmid-mediated expression of IκBγ-144-Rz produced a significant inhibition of the metastatic progression of B16-F10 cells to the lung and resulted in significant anti-invasive and pro-apoptotic effects on murine Lewis lung carcinoma cells. Our results suggest a novel role for IκBγ in promoting the metastatic progression of melanoma. PMID:19179607

  15. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  16. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  17. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  18. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  19. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  20. 30 CFR 18.43 - Explosion-proof splice boxes.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Explosion-proof splice boxes. 18.43 Section 18... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and adequately insulated. Strain clamps shall be provided for all cables entering a splice box....

  1. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  2. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  3. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  4. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  5. Schizophyllum commune has an extensive and functional alternative splicing repertoire

    PubMed Central

    Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

    2016-01-01

    Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically. PMID:27659065

  6. Splicing biomarkers of disease severity in myotonic dystrophy

    PubMed Central

    Nakamori, Masayuki; Sobczak, Krzysztof; Puwanant, Araya; Welle, Steve; Eichinger, Katy; Pandya, Shree; Dekdebrun, Jeannne; Heatwole, Chad R.; McDermott, Michael P.; Chen, Tian; Cline, Melissa; Tawil, Rabi; Osborne, Robert J.; Wheeler, Thurman M.; Swanson, Maurice; Moxley, Richard T.; Thornton, Charles A.

    2014-01-01

    Objective To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2). Methods In a discovery cohort we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. Results Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue. Interpretation Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy. PMID:23929620

  7. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    SciTech Connect

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  8. Inhibition of Splicing but not Cleavage at the 5' Splice Site by Truncating Human β -globin Pre-mRNA

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

    Human β -globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.

  9. Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts.

    PubMed

    Putnam, W C; Daniher, A T; Trawick, B N; Bashkin, J K

    2001-05-15

    Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phenanthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.

  10. Analyses of kinetic solvent isotope effects of a hammerhead ribozyme reaction in NH4+ and Li+ ions.

    PubMed

    Takagi, Yasuomi; Taira, Kazunari

    2002-01-01

    Hammerhead ribozymes have been considered to be divalent-metalloenzymes. However, this was recently questioned by the finding that the reaction can proceed without any divalent metal ions in the presence of high concentrations of monovalent ions such as NH4+/Li+ ions. Thus, one might think divalent metal ions are not involved in chemical step in the catalytic mechanism. To investigate the involvement of the monovalent ions, we analyzed the deuterium solvent isotope effects in the reactions. Our present analysis indicates a proton transfer(s) occurs only in the NH(4+)-mediated reaction, not in the Li(+)-mediated. Most simple interpretation is that NH4+ works as a general acid catalyst and Li+ as Lewis acid catalyst. This suggests hammerhead ribozymes can change catalyst upon their surrounding conditions.

  11. Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis

    PubMed Central

    Schommartz, Tim; Loroch, Stefan; Alawi, Malik; Grundhoff, Adam; Sickmann, Albert

    2016-01-01

    ABSTRACT Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. IMPORTANCE Herpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when

  12. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  13. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs. PMID:27606339

  14. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs.

  15. RNA structure in splicing: An evolutionary perspective.

    PubMed

    Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G

    2016-09-01

    Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements. PMID:27454491

  16. Chemical Feasibility of the General Acid/Base Mechanism of glmS Ribozyme Self-Cleavage

    PubMed Central

    Dubecký, Matúš; Walter, Nils G.; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

    2015-01-01

    In numerous Gram-positive bacteria, the glmS ribozyme or catalytic riboswitch regulates the expression of glucosamine-6-phosphate (GlcN6P) synthase via site-specific cleavage of its sugar-phosphate backbone in response to GlcN6P ligand binding. Biochemical data have suggested a crucial catalytic role for an active site guanine (G40 in Thermoanaerobacter tengcongensis, G33 in Bacillus anthracis). We used hybrid quantum chemical/molecular mechanical (QM/MM) calculations to probe the mechanism where G40 is deprotonated and acts as a general base. The calculations suggest that the deprotonated guanine G40− is sufficiently reactive to overcome the thermodynamic penalty arising from its rare protonation state, and thus is able to activate the A-1(2’-OH) group toward nucleophilic attack on the adjacent backbone. Furthermore, deprotonation of A-1(2’-OH) and nucleophilic attack are predicted to occur as separate steps, where activation of A-1(2’-OH) precedes nucleophilic attack. Conversely, the transition state associated with the rate-determining step corresponds to concurrent nucleophilic attack and protonation of the G1(O5’) leaving group by the ammonium moiety of the GlcN6P cofactor. Overall, our calculations help to explain the crucial roles of G40 (as a general base) and GlcN6P (as a general acid) during glmS ribozyme self-cleavage. In addition, we show that the QM/MM description of the glmS ribozyme self-cleavage reaction is significantly more sensitive to the size of the QM region and the quality of the QM-MM coupling than that of other small ribozymes. PMID:25858644

  17. Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells

    PubMed Central

    2012-01-01

    Background Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs. Findings Ribozymes, loaded on a cationic polymer-based vehicle, were delivered into cultured VSMCs. A significant impairment of the activation mechanisms triggered by PDGF-BB was demonstrated since cell migration decreased after treatments. In order to functionally validate the effects of PDGFR-β partial knock down we focused on the phosphorylation status of two proteins, protein disulfide isomerase-A3 (PDI-A3) and heat shock protein-60 (HSP-60), previously identified as indicative of VSMC phenotypic switch after PDGF-BB stimulation. Interestingly, while PDI-A3 phosphorylation was counteracted by the ribozyme administration indicating that PDI-A3 is a factor downstream the receptor signalling cascade, the HSP-60 phosphorylation status was greatly increased by the ribozyme administration. Conclusion These contradictory observations suggested that PDGF-BB might trigger different parallel pathways that could be modulated by alternative isoforms of the receptors for the growth factor. In conclusion the knock down strategy here described enables to discriminate between two tightly intermingled pathways. Moreover it opens new attractive perspectives in functional investigations where combined gene knock down and proteomic technologies would allow the identification of key factors and pathways involved in VSMC-linked pathological disorders. PMID:22676333

  18. A Single-Stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme

    PubMed Central

    Nguyen, Phuong; Shi, Xuesong; Sigurdsson, Snorri Th.; Herschlag, Daniel

    2013-01-01

    Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes. PMID:23900919

  19. Chemical feasibility of the general acid/base mechanism of glmS ribozyme self-cleavage.

    PubMed

    Dubecký, Matúš; Walter, Nils G; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

    2015-10-01

    In numerous Gram-positive bacteria, the glmS ribozyme or catalytic riboswitch regulates the expression of glucosamine-6-phosphate (GlcN6P) synthase via site-specific cleavage of its sugar-phosphate backbone in response to GlcN6P ligand binding. Biochemical data have suggested a crucial catalytic role for an active site guanine (G40 in Thermoanaerobacter tengcongensis, G33 in Bacillus anthracis). We used hybrid quantum chemical/molecular mechanical (QM/MM) calculations to probe the mechanism where G40 is deprotonated and acts as a general base. The calculations suggest that the deprotonated guanine G40(-) is sufficiently reactive to overcome the thermodynamic penalty arising from its rare protonation state, and thus is able to activate the A-1(2'-OH) group toward nucleophilic attack on the adjacent backbone. Furthermore, deprotonation of A-1(2'-OH) and nucleophilic attack are predicted to occur as separate steps, where activation of A-1(2'-OH) precedes nucleophilic attack. Conversely, the transition state associated with the rate-determining step corresponds to concurrent nucleophilic attack and protonation of the G1(O5') leaving group by the ammonium moiety of the GlcN6P cofactor. Overall, our calculations help to explain the crucial roles of G40 (as a general base) and GlcN6P (as a general acid) during glmS ribozyme self-cleavage. In addition, we show that the QM/MM description of the glmS ribozyme self-cleavage reaction is significantly more sensitive to the size of the QM region and the quality of the QM-MM coupling than that of other small ribozymes.

  20. The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site.

    PubMed Central

    Murray, J B; Adams, C J; Arnold, J R; Stockley, P G

    1995-01-01

    We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5' to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2'-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site. PMID:7487885

  1. Splicing-coupled 3' end formation requires a terminal splice acceptor site, but not intron excision.

    PubMed

    Davidson, Lee; West, Steven

    2013-08-01

    Splicing of human pre-mRNA is reciprocally coupled to 3' end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3' end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3' end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3' end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3' end formation, but that on-going intron removal is less critical. PMID:23716637

  2. The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing

    PubMed Central

    Du, Chen; Ma, Xinran; Meruvu, Sunitha; Hugendubler, Lynne; Mueller, Elisabetta

    2014-01-01

    Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPARγ in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638’s interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms. PMID:25024404

  3. Long-term Expression of Apolipoprotein B mRNA-specific Hammerhead Ribozyme via scAAV8.2 Vector Inhibits Atherosclerosis in Mice

    PubMed Central

    Nischal, Hersharan; Sun, Hua; Wang, Yuchun; Ford, David A; Cao, Ying; Wei, Peng; Teng, Ba-Bie

    2013-01-01

    Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development. We designed two hammerhead ribozymes targeted at the nucleotides of apoB mRNA GUC2326 (designated RB1) and GUA6679 (designated RB15), and we used self-complementary adeno-associated virus 8.2 (scAAV8.2) vector to deliver these active ribozymes of RB1, RB15, combination of RB1/RB15, and an inactive hammerhead ribozyme RB15 mutant to atherosclerosis-prone LDb mice (Ldlr−/−Apobec1−/−). LDb mice lack both low density lipoproteins (LDL) receptor (Ldlr−/−) and apoB mRNA editing enzyme (Apobec1−/−) genes and develop atherosclerosis spontaneously. After the RB1, RB15, or combination of RB1/RB15 ribozymes treatment, the LDb mice had significantly decreased plasma triglyceride and apoB levels, resulting in markedly decreased of atherosclerotic lesions, Furthermore, the active ribozymes treatment decreased the levels of diacylglycerol acyltransferase 1 (Dgat1) mRNA and the levels of multiple diacylglycerol (DAG) molecular species. These results provide the first evidence that decreased apoB levels results to reduction of Dgat1 expression and triglyceride levels (TAG), which had a significant impact on the development of atherosclerosis. PMID:24084845

  4. A Rearrangement of the Guanosine-Binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site†

    PubMed Central

    Forconi, Marcello; Sengupta, Raghuvir N.; Piccirilli, Joseph A.; Herschlag, Daniel

    2010-01-01

    Protein enzymes appear to use extensive packing and hydrogen-bonding interactions to precisely position catalytic groups within active sites. Due to their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face additional challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously determined that the hydrogen bond between the exocyclic amino group of guanosine and the 2′-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double-mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated to other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chemical transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site. PMID:20175542

  5. Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

    PubMed Central

    Biondi, Elisa; Poudyal, Raghav R.; Forgy, Joshua C.; Sawyer, Andrew W.; Maxwell, Adam W. R.; Burke, Donald H.

    2013-01-01

    The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu2+ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu2+, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu2+ binding by RNA; rather, these data establish that Cu2+ enters the active site within a Cu2+•GTPγS or Cu2+•GTP chelation complex, and that Cu2+•nucleobase interactions further enforce Cu2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg2+ coordination is structural rather than catalytic. Replacing Mg2+ with alkaline earths of increasing ionic radii (Ca2+, Sr2+ and Ba2+) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step. PMID:23358821

  6. Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment

    PubMed Central

    Biondi, Elisa; Branciamore, Sergio; Maurel, Marie-Christine; Gallori, Enzo

    2007-01-01

    Background The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis. Results The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay. Conclusion These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization. PMID:17767730

  7. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan T; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz. PMID:25800735

  8. SplicePie: a novel analytical approach for the detection of alternative, non-sequential and recursive splicing.

    PubMed

    Pulyakhina, Irina; Gazzoli, Isabella; 't Hoen, Peter A C; Verwey, Nisha; den Dunnen, Johan T; den Dunnen, Johan; Aartsma-Rus, Annemieke; Laros, Jeroen F J

    2015-07-13

    Alternative splicing is a powerful mechanism present in eukaryotic cells to obtain a wide range of transcripts and protein isoforms from a relatively small number of genes. The mechanisms regulating (alternative) splicing and the paradigm of consecutive splicing have recently been challenged, especially for genes with a large number of introns. RNA-Seq, a powerful technology using deep sequencing in order to determine transcript structure and expression levels, is usually performed on mature mRNA, therefore not allowing detailed analysis of splicing progression. Sequencing pre-mRNA at different stages of splicing potentially provides insight into mRNA maturation. Although the number of tools that analyze total and cytoplasmic RNA in order to elucidate the transcriptome composition is rapidly growing, there are no tools specifically designed for the analysis of nuclear RNA (which contains mixtures of pre- and mature mRNA). We developed dedicated algorithms to investigate the splicing process. In this paper, we present a new classification of RNA-Seq reads based on three major stages of splicing: pre-, intermediate- and post-splicing. Applying this novel classification we demonstrate the possibility to analyze the order of splicing. Furthermore, we uncover the potential to investigate the multi-step nature of splicing, assessing various types of recursive splicing events. We provide the data that gives biological insight into the order of splicing, show that non-sequential splicing of certain introns is reproducible and coinciding in multiple cell lines. We validated our observations with independent experimental technologies and showed the reliability of our method. The pipeline, named SplicePie, is freely available at: https://github.com/pulyakhina/splicing_analysis_pipeline. The example data can be found at: https://barmsijs.lumc.nl/HG/irina/example_data.tar.gz.

  9. Functional analysis of a C. elegans trans-splice acceptor.

    PubMed Central

    Conrad, R; Liou, R F; Blumenthal, T

    1993-01-01

    The rol-6 gene is trans-spliced to the 22 nt leader, SL1, 173 nt downstream of the transcription start. We have analyzed splicing in transformants carrying extrachromosomal arrays of rol-6 with mutations in the trans-splice acceptor site. This site is a close match to the consensus, UUUCAG, that is highly conserved in both trans-splice and intron acceptor sites in C. elegans. When the trans-splice site was inactivated by mutating the perfectly-conserved AG, trans-splicing still occurred, but at a cryptic site 20 nt upstream. We tested the frequency with which splicing switched from the normal site to the cryptic site when the pyrimidines at this site were changed to A's. Since most C. elegans 3' splice sites lack an obvious polypyrimidine tract, we hypothesized that these four pyrimidines might play this role, and indeed mutation of these bases caused splicing to switch to the cryptic site. We also demonstrated that a major reason the downstream site is normally favored is because it occurs at a boundary between A+U rich and non-A+U rich RNA. When the RNA between the two splice sites was made less A+U rich, splicing occurred preferentially at the upstream site. Images PMID:8451190

  10. Deregulation of splicing factors and breast cancer development.

    PubMed

    Silipo, Marco; Gautrey, Hannah; Tyson-Capper, Alison

    2015-10-01

    It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single-nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.

  11. An alternative splicing program promotes adipose tissue thermogenesis.

    PubMed

    Vernia, Santiago; Edwards, Yvonne Jk; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

    2016-01-01

    Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. PMID:27635635

  12. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  13. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  14. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site.

    PubMed

    Mueller, Nancy; Berkhout, Ben; Das, Atze T

    2015-07-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins. PMID:25779589

  15. Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

    PubMed Central

    2010-01-01

    Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies. PMID:20525254

  16. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    NASA Astrophysics Data System (ADS)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  17. Sequence-specific flexibility organization of splicing flanking sequence and prediction of splice sites in the human genome.

    PubMed

    Zuo, Yongchun; Zhang, Pengfei; Liu, Li; Li, Tao; Peng, Yong; Li, Guangpeng; Li, Qianzhong

    2014-09-01

    More and more reported results of nucleosome positioning and histone modifications showed that DNA structure play a well-established role in splicing. In this study, a set of DNA geometric flexibility parameters originated from molecular dynamics (MD) simulations were introduced to discuss the structure organization around splice sites at the DNA level. The obtained profiles of specific flexibility/stiffness around splice sites indicated that the DNA physical-geometry deformation could be used as an alternative way to describe the splicing junction region. In combination with structural flexibility as discriminatory parameter, we developed a hybrid computational model for predicting potential splicing sites. And the better prediction performance was achieved when the benchmark dataset evaluated. Our results showed that the mechanical deformability character of a splice junction is closely correlated with both the splice site strength and structural information in its flanking sequences.

  18. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H+ in the active site, which acts as the general acid, and a partially hydrated Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2′-OH) nucleophile and the nucleophilic attack of the resulting U-1(2′-O−) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  19. The influence of Argonaute proteins on alternative RNA splicing.

    PubMed

    Batsché, Eric; Ameyar-Zazoua, Maya

    2015-01-01

    Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO.

  20. Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

    PubMed Central

    Jiménez-López, Claudia; Lorenz, Michael C.; van Hoof, Ambro

    2013-01-01

    Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes. PMID:23516382

  1. Evolution of Nova-Dependent Splicing Regulation in the Brain

    PubMed Central

    Živin, Marko; Darnell, Robert B

    2007-01-01

    A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

  2. Anti-tumor activity of splice-switching oligonucleotides

    PubMed Central

    Bauman, John A.; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard

    2010-01-01

    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing. PMID:20719743

  3. Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

    PubMed

    Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

    2005-06-01

    In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed

  4. Entropic contributions to the splicing process

    NASA Astrophysics Data System (ADS)

    Osella, Matteo; Caselle, Michele

    2009-12-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model.

  5. Distal regulation of alternative splicing by splicing enhancer in equine beta-casein intron 1.

    PubMed

    Lenasi, Tina; Peterlin, B Matija; Dovc, Peter

    2006-03-01

    The complexity of cotranscriptional splicing is reflected in the coordinated interplay between various cis-elements and transacting factors. In this report, we demonstrated that a cis-element in intron 1 of the equine beta-casein gene (intronic splicing enhancer 1, ISE1) increases the inclusion of all weak exons in its pre-mRNA. The ISE1 also functioned on a hybrid transcript, which was transcribed from the alpha-globin promoter, where it increased the inclusion of the human fibronectin EDA exon and the beta-casein exon 5. The region of ISE1 necessary for its function included the same sequence as is found in some exonic splicing enhancers. Since the ISE1 influenced the splicing of the entire transcript from intron 1, we propose a model for the cotranscriptional splicing of beta-casein mRNA, where the 5' end of the growing transcript remains associated with the C-terminal domain of RNA polymerase II. Thus, the ISE1 remains in close proximity to the mRNA exit groove throughout transcription and influences all weak exons as soon as they are copied.

  6. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    PubMed Central

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-01-01

    Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy. PMID:26325627

  7. Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5' splice site to the internal branch acceptor site.

    PubMed Central

    Köhrer, K; Domdey, H

    1988-01-01

    Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing. Images PMID:3054807

  8. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  9. Splicing variants of porcine synphilin-1.

    PubMed

    Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian

    2015-09-01

    Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation. PMID:26101749

  10. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

  11. The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

    PubMed

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

    The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

  12. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

  13. The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events

    PubMed Central

    Warzecha, Claude C.; Shen, Shihao; Xing, Yi; Carstens, Russ P.

    2010-01-01

    Cell-type and tissue-specific alternative splicing events are regulated by combinatorial control involving both abundant RNA binding proteins as well as those with more discrete expression and specialized functions. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are recently discovered epithelial-specific RNA binding proteins that promote splicing of the epithelial variant of the FGFR2, ENAH, CD44 and CTNND1 transcripts. To catalogue a larger set of splicing events under the regulation of the ESRPs we profiled splicing changes induced by RNA interference-mediated knockdown of ESRP1 and ESRP2 expression in a human epithelial cell line using the splicing sensitive Affymetrix Exon ST1.0 Arrays. Analysis of the microarray data resulted in the identification of over a hundred candidate ESRP regulated splicing events. We were able to independently validate 38 of these targets by RT-PCR. The ESRP regulated events encompass all known types of alternative splicing events, most prominent being alternative cassette exons and splicing events leading to alternative 3' terminal exons. Importantly, a number of these regulated splicing events occur in gene transcripts that encode proteins with well-described roles in the regulation of actin cytoskeleton organization, cell-cell adhesion, cell polarity and cell migration. In sum, this work reveals a novel list of transcripts differentially spliced in epithelial and mesenchymal cells, implying that coordinated alternative splicing plays a critical role in determination of cell type identity. These results further establish ESRP1 and ESRP2 as global regulators of an epithelial splicing regulatory network. PMID:19829082

  14. Half pint/Puf68 is required for negative regulation of splicing by the SR splicing factor Transformer2.

    PubMed

    Wang, Shanzhi; Wagner, Eric J; Mattox, William

    2013-08-01

    The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion. PMID:23880637

  15. Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors.

    PubMed

    Scognamiglio, Viviana; Antonacci, Amina; Lambreva, Maya D; Litescu, Simona C; Rea, Giuseppina

    2015-12-15

    Biosensors are powerful tunable systems able to switch between an ON/OFF status in response to an external stimulus. This extraordinary property could be engineered by adopting synthetic biology or biomimetic chemistry to obtain tailor-made biosensors having the desired requirements of robustness, sensitivity and detection range. Recent advances in both disciplines, in fact, allow to re-design the configuration of the sensing elements - either by modifying toggle switches and gene networks, or by producing synthetic entities mimicking key properties of natural molecules. The present review considered the role of synthetic biology in sustaining biosensor technology, reporting examples from the literature and reflecting on the features that make it a useful tool for designing and constructing engineered biological systems for sensing application. Besides, a section dedicated to bioinspired synthetic molecules as powerful tools to enhance biosensor potential is reported, and treated as an extension of the concept of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules. Thus, the design of synthetic molecules, such as aptamers, biomimetics, molecular imprinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic chemistry. PMID:26277908

  16. Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors.

    PubMed

    Scognamiglio, Viviana; Antonacci, Amina; Lambreva, Maya D; Litescu, Simona C; Rea, Giuseppina

    2015-12-15

    Biosensors are powerful tunable systems able to switch between an ON/OFF status in response to an external stimulus. This extraordinary property could be engineered by adopting synthetic biology or biomimetic chemistry to obtain tailor-made biosensors having the desired requirements of robustness, sensitivity and detection range. Recent advances in both disciplines, in fact, allow to re-design the configuration of the sensing elements - either by modifying toggle switches and gene networks, or by producing synthetic entities mimicking key properties of natural molecules. The present review considered the role of synthetic biology in sustaining biosensor technology, reporting examples from the literature and reflecting on the features that make it a useful tool for designing and constructing engineered biological systems for sensing application. Besides, a section dedicated to bioinspired synthetic molecules as powerful tools to enhance biosensor potential is reported, and treated as an extension of the concept of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules. Thus, the design of synthetic molecules, such as aptamers, biomimetics, molecular imprinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic chemistry.

  17. SR protein kinases promote splicing of nonconsensus introns.

    PubMed

    Lipp, Jesse J; Marvin, Michael C; Shokat, Kevan M; Guthrie, Christine

    2015-08-01

    Phosphorylation of the spliceosome is essential for RNA splicing, yet how and to what extent kinase signaling affects splicing have not been defined on a genome-wide basis. Using a chemical genetic approach, we show in Schizosaccharomyces pombe that the SR protein kinase Dsk1 is required for efficient splicing of introns with suboptimal splice sites. Systematic substrate mapping in fission yeast and human cells revealed that SRPKs target evolutionarily conserved spliceosomal proteins, including the branchpoint-binding protein Bpb1 (SF1 in humans), by using an RXXSP consensus motif for substrate recognition. Phosphorylation of SF1 increases SF1 binding to introns with nonconsensus splice sites in vitro, and mutation of such sites to consensus relieves the requirement for Dsk1 and phosphorylated Bpb1 in vivo. Modulation of splicing efficiency through kinase signaling pathways may allow tuning of gene expression in response to environmental and developmental cues. PMID:26167880

  18. Dysfunctional Gene Splicing as a Potential Contributor to Neuropsychiatric Disorders

    PubMed Central

    Glatt, Stephen J.; Cohen, Ori S.; Faraone, Stephen V.; Tsuang, Ming T.

    2011-01-01

    Alternative pre-mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of “gene-focused” epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders. PMID:21438146

  19. Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis.

    PubMed Central

    Shan, Shu-ou; Herschlag, Daniel

    2002-01-01

    Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2'-OH of G. These results suggest that the energy from the metal ion substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous-work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis. PMID:12166641

  20. Folding of the Tetrahymena Ribozyme by Polyamines: Importance of Counterion Valence and Size

    SciTech Connect

    Koculi,E.; Lee, N.; Thirumalai, D.; Woodson, S.

    2004-01-01

    Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg{sup 2+}. The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.

  1. Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogs for the glmS Ribozyme

    PubMed Central

    Posakony, Jeffrey J.; Ferré-D'Amaré, Adrian R.

    2013-01-01

    Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactor of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogs, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz) and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogs. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. PMID:23578404

  2. Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

    PubMed

    Petrie, Katherine L; Joyce, Gerald F

    2014-10-01

    The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

  3. Ligation of the hairpin ribozyme in cis induced by freezing and dehydration

    PubMed Central

    KAZAKOV, SERGEI A.; BALATSKAYA, SVETLANA V.; JOHNSTON, BRIAN H.

    2006-01-01

    Although reducing the temperature slows most chemical reactions, freezing can stimulate some reactions by mechanisms that are only partially understood. Here we show that freezing stimulates the self-ligation (circularization) of linear forms of the hairpin ribozyme (HPR) containing 2′,3′-cyclic phosphate and 5′-OH termini. Divalent metal ions (M2+) are not required, but monovalent cations and anions at millimolar concentrations can have various effects on this reaction depending on the specific ion. Under optimal conditions, the observed rate of M2+-independent self-ligation reaches a peak (0.04 min−1) at −10°C with a yield of −60% after 1 h. In contrast, no ligation occurs either at above 0°C or in solutions that remain unfrozen when supercooled to subzero temperatures. Under freezing conditions, the cleavage–ligation equilibrium strongly favors ligation. Besides freezing, evaporation of the aqueous solvent as well as the presence of ethanol at levels of 40% or above can also induce M2+-independent HPR ligation at 25°C. We argue that partial RNA dehydration, which is a common feature of freezing, evaporation, and the presence of ethanol, is a key factor supporting HPR ligation activity at both above- and below-freezing temperatures. In the context of the RNA world hypothesis, freezing-induced ligation is an attractive mechanism by which complex RNAs could have evolved under conditions in which RNA was relatively protected against degradation. PMID:16495237

  4. Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor.

    PubMed Central

    Murray, J B; Arnold, J R

    1996-01-01

    A screening of a range of common laboratory antibiotics for inhibition of the hammerhead ribozyme has shown that in addition to certain aminoglycosides (most notably neomycin B) the tetracyclines are also effective inhibitors, with chlorotetracycline being more effective than tetracycline. Inhibition by chlorotetracycline is not as strong as that by neomycin B but is more complicated, with at least two binding sites apparent. As with hammerhead inhibition by neomycin B, chlorotetracycline inhibition can be overcome by raising the concentration of the Mg2+ ion cofactor. We find that around six Mg2+ ions will displace neomycin B, compared with twelve for chlorotetracycline. Inhibition observed in the presence of mixtures of neomycin B and chlorotetracycline is consistent with separate binding sites on the hammerhead for these two classes of antibiotic. Under certain conditions of the mixing order and low concentration of chlorotetracycline, enhancement of single-turnover hammerhead cleavage by up to 20% is observed, with higher concentrations of antibiotic being inhibitory. We have also found that the presence of 2.5% (v/v) DMSO causes a 30% enhancement of the single-turnover cleavage. These results thus extend the range of known inhibitors of hammerhead cleavage, and also demonstrate how the cleavage can be accelerated. PMID:8760373

  5. Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

    PubMed Central

    Lee, Tai-Sung; Wong, Kin-Yiu; Giambasu, George M.; York, Darrin M.

    2016-01-01

    Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis. PMID:24156941

  6. CHARMM force field parameters for simulation of reactive intermediates in native and thio-substituted ribozymes.

    PubMed

    Mayaan, Evelyn; Moser, Adam; MacKerell, Alexander D; York, Darrin M

    2007-01-30

    Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations, in a fashion consistent with the CHARMM27 all-atom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg(2+) and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)// B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges. Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle, and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the nonstandard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.

  7. In vitro evolution of distinct self-cleaving ribozymes in diverse environments

    PubMed Central

    Popović, Milena; Fliss, Palmer S.; Ditzler, Mark A.

    2015-01-01

    In vitro evolution experiments have long been used to evaluate the roles of RNA in both modern and ancient biology, and as a tool for biotechnology applications. The conditions under which these experiments have been conducted, however, do not reflect the range of cellular environments in modern biology or our understanding of chemical environments on the early earth, when the atmosphere and oceans were largely anoxic and soluble Fe2+ was abundant. To test the impact of environmental factors relevant to RNA's potential role in the earliest forms of life, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the presence of either Fe2+ or Mg2+. Populations evolved under these different conditions are dominated by different sequences and secondary structures, demonstrating global differences in the underlying fitness landscapes. Comparisons between evolutionary outcomes and catalytic activities also indicate that Mg2+ can readily take the place of Fe2+ in supporting the catalysis of RNA cleavage at neutral pH, but not at lower pH. These results highlight the importance of considering the specific environments in which functional biopolymers evolve when evaluating their potential roles in the origin of life, extant biology, or biotechnology. PMID:26130717

  8. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data.

    PubMed

    Kroll, Jose E; Kim, Jihoon; Ohno-Machado, Lucila; de Souza, Sandro J

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background. Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files) using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events. Availability and Implementation. Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress.

  9. Superconducting cable-in-conduit low resistance splice

    DOEpatents

    Artman, Thomas A.

    2003-06-24

    A low resistance splice connects two cable-in-conduit superconductors to each other. Dividing collars for arranging sub-cable units from each conduit are provided, along with clamping collars for mating each sub-cable wire assembly to form mated assemblies. The mated assemblies ideally can be accomplished by way of splicing collar. The mated assemblies are cooled by way of a flow of coolant, preferably helium. A method for implementing such a splicing is also described.

  10. A Comparison of Vanadate to a 2'-5' Linkage at the Active Site of a Small Ribozyme Suggests a Role for Water in Transition-State Stabilization

    SciTech Connect

    Torelli, A.T.; Krucinska, J.; Wedekind, J.E.

    2009-06-04

    The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.

  11. Phylogenetic footprinting of non-coding RNA: hammerhead ribozyme sequences in a satellite DNA family of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae)

    PubMed Central

    2010-01-01

    Background The great variety in sequence, length, complexity, and abundance of satellite DNA has made it difficult to ascribe any function to this genome component. Recent studies have shown that satellite DNA can be transcribed and be involved in regulation of chromatin structure and gene expression. Some satellite DNAs, such as the pDo500 sequence family in Dolichopoda cave crickets, have a catalytic hammerhead (HH) ribozyme structure and activity embedded within each repeat. Results We assessed the phylogenetic footprints of the HH ribozyme within the pDo500 sequences from 38 different populations representing 12 species of Dolichopoda. The HH region was significantly more conserved than the non-hammerhead (NHH) region of the pDo500 repeat. In addition, stems were more conserved than loops. In stems, several compensatory mutations were detected that maintain base pairing. The core region of the HH ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among 198 sequences. RNA folding of the HH sequences revealed that a potentially active HH ribozyme can be found in most of the Dolichopoda populations and species. Conclusions The phylogenetic footprints suggest that the HH region of the pDo500 sequence family is selected for function in Dolichopoda cave crickets. However, the functional role of HH ribozymes in eukaryotic organisms is unclear. The possible functions have been related to trans cleavage of an RNA target by a ribonucleoprotein and regulation of gene expression. Whether the HH ribozyme in Dolichopoda is involved in similar functions remains to be investigated. Future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead. PMID:20047671

  12. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. PMID:26846640

  13. Viral interactions with components of the splicing machinery.

    PubMed

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses.

  14. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease.

  15. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  16. Some characteristics of probabilistic one-sided splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Fong, Wan Heng; Sarmin, Nor Haniza; Turaev, Sherzod

    2013-04-01

    A theoretical model for DNA computing using the recombination behavior of DNA molecules known as asplicing system has been introduced in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings at the specific places and attaches the prefix of the first string to the suffix of the second string and the prefix of the second string to the suffix of the first string yielding the new strings. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions for splicing systems have been considered to increase the computational power of the languages generated. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic one-sided splicing systems, which are special types of probabilistic splicing systems, are investigated. We prove that probabilistic one-sided splicing systems can also increase the computational power of the languages generated.

  17. Evolutionary Insights into RNA trans-Splicing in Vertebrates

    PubMed Central

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-01-01

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

  18. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

    PubMed Central

    2010-01-01

    Background Genome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much smaller than observed in

  19. Viral interactions with components of the splicing machinery.

    PubMed

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses. PMID:27571697

  20. Age-dependent gain of alternative splice forms and biased duplication explain the relation between splicing and duplication

    PubMed Central

    Roux, Julien; Robinson-Rechavi, Marc

    2011-01-01

    We analyze here the relation between alternative splicing and gene duplication in light of recent genomic data, with a focus on the human genome. We show that the previously reported negative correlation between level of alternative splicing and family size no longer holds true. We clarify this pattern and show that it is sufficiently explained by two factors. First, genes progressively gain new splice variants with time. The gain is consistent with a selectively relaxed regime, until purifying selection slows it down as aging genes accumulate a large number of variants. Second, we show that duplication does not lead to a loss of splice forms, but rather that genes with low levels of alternative splicing tend to duplicate more frequently. This leads us to reconsider the role of alternative splicing in duplicate retention. PMID:21173032

  1. Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P

    PubMed Central

    2014-01-01

    Background Hepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide. However, HCV resistance to interferon treatment is one of the important clinical implications, suggesting the necessity to seek new therapies. It has already been shown that some forms of the catalytic RNA moiety from E. coli RNase P, M1 RNA, can be introduced into the cytoplasm of mammalian cells for the purpose of carrying out targeted cleavage of mRNA molecules. Our study is to use an engineering M1 RNA (i.e. M1GS) for inhibiting HCV replication and demonstrates the utility of this ribozyme for antiviral applications. Results By analyzing the sequence and structure of the 5′ untranslated region of HCV RNA, a putative cleavage site (C67-G68) was selected for ribozyme designing. Based on the flanking sequence of this site, a targeting M1GS ribozyme (M1GS-HCV/C67) was constructed by linking a custom guide sequence (GS) to the 3′ termini of catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli through an 88 nt-long bridge sequence. In vitro cleavage assays confirmed that the engineered M1GS ribozyme cleaved the targeted RNA specifically. Moreover, ~85% reduction in the expression levels of HCV proteins and >1000-fold reduction in viral growth were observed in supernatant of cultured cells that transfected the functional ribozyme. In contrast, the HCV core expression and viral growth were not significantly affected by a “disabled” ribozyme (i.e. M1GS-HCV/C67*). Moreover, cholesterol-conjugated M1GS ribozyme (i.e. Chol-M1GS-HCV/C67) showed almost the same bioactivities with M1GS-HCV/C67, demonstrating the potential to improve in vivo pharmacokinetic properties of M1GS-based RNA therapeutics. Conclusion Our results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV. PMID:24885776

  2. Plant synthetic biology.

    PubMed

    Liu, Wusheng; Stewart, C Neal

    2015-05-01

    Plant synthetic biology is an emerging field that combines engineering principles with plant biology toward the design and production of new devices. This emerging field should play an important role in future agriculture for traditional crop improvement, but also in enabling novel bioproduction in plants. In this review we discuss the design cycles of synthetic biology as well as key engineering principles, genetic parts, and computational tools that can be utilized in plant synthetic biology. Some pioneering examples are offered as a demonstration of how synthetic biology can be used to modify plants for specific purposes. These include synthetic sensors, synthetic metabolic pathways, and synthetic genomes. We also speculate about the future of synthetic biology of plants.

  3. Tissue-specific alternative splicing of Shaker potassium channel transcripts results from distinct modes of regulating 3' splice choice.

    PubMed

    Iverson, L E; Mottes, J R; Yeager, S A; Germeraad, S E

    1997-05-01

    Alternative splicing of precursor RNA enables a single gene to encode multiple protein isoforms with different functional characteristics and tissue distributions. Differential splicing of Drosophila Shaker (Sh) gene transcripts regulates the tissue-specific expression of kinetically distinct potassium ion channels throughout development. Regulation of Sh alternative splicing is being examined in germline transformants using lacZ as a reporter gene. P-element constructs were generated in which one or both of the two mutually exclusive Sh 3' acceptor sites were positioned in the same translational reading frame as the lacZ coding sequences. The constructs were introduced into the germline and the transgenic animals examined for tissue-specific beta-galactosidase expression patterns. Some tissues exhibit "promiscuous" splicing; these tissues are competent to splice to either 3' acceptor even when both are present on the same pre-mRNA. In other tissues splice choice results from competition between the two 3' sites; these tissues can splice to either site when it is the only available 3' acceptor, but when given a choice will splice to only one of the two 3' acceptors. In some tissues, splicing occurs exclusively at only one of the 3' acceptor sites; these tissues are not competent to splice to one of the sites even if it is the only 3' acceptor present on the pre-mRNA. These results suggests that multiple, distinct regulatory modes are operating to control tissue-specific alternative splicing of Sh 3' domains and are discussed in terms of potential underlying mechanisms for regulating the tissue-specific expression of alternatively spliced genes.

  4. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

    PubMed

    Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

  5. Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

    PubMed Central

    KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

    2005-01-01

    Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

  6. The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

    PubMed Central

    Bassi, G S; Murchie, A I; Lilley, D M

    1996-01-01

    The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. PMID:8752086

  7. A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme

    PubMed Central

    Yoshinari, Koichi; Taira, Kazunari

    2000-01-01

    We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thiosubstituted substrates S11SpS and S11RpS, in which the respective pro-Sp and pro-Rp oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg2+ or Ca2+ ions the rate of ribozyme-catalyzed cleavage of S11SpS was as high as that of S11O, whereas the corresponding rate for S11RpS was nearly four orders of magnitude lower than that for either S11O or S11SpS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd2+ ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the Rp position of the cleavage site with the added Cd2+ ion. However, our present analysis demonstrates that (i) the added Cd2+ ion binds at the P9 site; (ii) the bound Cd2+ ion at the P9 site replaces two Mg2+ or two Ca2+ ions, an observation that suggests a different mode of interaction with the added Cd2+ ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd2+ ion does not interact with the sulfur atom at the Rp position of the scissile phosphate either in the ground state or in the transition state. PMID:10734192

  8. Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme.

    PubMed

    Smith, M Duane; Collins, Richard A

    2007-04-01

    A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec(-1), similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pK(a)s of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D(2)O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH- and D(2)O-independent cleavage approximately 10(3)-fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate approximately 10(3)-fold, but the A756C mutant retains pH- and D(2)O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.

  9. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  10. [From synthetic biology to synthetic humankind].

    PubMed

    Nouvel, Pascal

    2015-01-01

    In this paper, we propose an historical survey of the expression "synthetic biology" in order to identify its main philosophical components. The result of the analysis is then used to investigate the meaning of the notion of "synthetic man". It is shown that both notions share a common philosophical background that can be summed up by the short but meaningful assertion: "biology is technology". The analysis allows us to distinguish two notions that are often confused in transhumanist literature: the notion of synthetic man and the notion of renewed man. The consequences of this crucial distinction are discussed.

  11. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

    PubMed Central

    Berkers, Celia R.; de Jong, Annemieke; Schuurman, Karianne G.; Linnemann, Carsten; Meiring, Hugo D.; Janssen, Lennert; Neefjes, Jacques J.; Schumacher, Ton N. M.; Rodenko, Boris

    2015-01-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags. PMID:26401003

  12. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    PubMed Central

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-01-01

    Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

  13. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

    PubMed

    Berkers, Celia R; de Jong, Annemieke; Schuurman, Karianne G; Linnemann, Carsten; Meiring, Hugo D; Janssen, Lennert; Neefjes, Jacques J; Schumacher, Ton N M; Rodenko, Boris; Ovaa, Huib

    2015-11-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

  14. Synthesis and Purification of a Hammerhead Ribozyme and a Fluorescein-Labeled RNA Substrate. A Biochemistry Laboratory: Part 1

    NASA Astrophysics Data System (ADS)

    Chow, Christine S.; Somne, Smita

    1999-05-01

    The applications of in vitro transcription and chemical synthesis of RNA are discussed. This laboratory describes the in vitro synthesis of a 38-nucleotide hammerhead ribozyme and the synthesis of a 17-nucleotide fluorescein-labeled RNA substrate by using standard phosphoramidite methodologies, two widely used methods in modern RNA research. The synthesis and purification procedures outlined allow students to develop an understanding of RNA handling procedures, synthesis of modified nucleic acids, gel electrophoresis, visualization of RNA by nonradioactive techniques, and quantitation of nucleic acids. The RNAs that are synthesized have applications in biotechnology and medicine; thus the students gain access to current problems in chemical and clinical research.

  15. Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity.

    PubMed

    Horton, T E; Clardy, D R; DeRose, V J

    1998-12-22

    Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These

  16. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    PubMed Central

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Geneviève; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; Østergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I

    2015-01-01

    Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing. PMID:26125040

  17. An artificial riboswitch for controlling pre-mRNA splicing.

    PubMed

    Kim, Dong-Suk; Gusti, Veronica; Pillai, Sailesh G; Gaur, Rajesh K

    2005-11-01

    Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant. PMID:16244133

  18. Evolution of alternative splicing in primate brain transcriptomes

    PubMed Central

    Lin, Lan; Shen, Shihao; Jiang, Peng; Sato, Seiko; Davidson, Beverly L.; Xing, Yi

    2010-01-01

    Alternative splicing is a predominant form of gene regulation in higher eukaryotes. The evolution of alternative splicing provides an important mechanism for the acquisition of novel gene functions. In this work, we carried out a genome-wide phylogenetic survey of lineage-specific splicing patterns in the primate brain, via high-density exon junction array profiling of brain transcriptomes of humans, chimpanzees and rhesus macaques. We identified 509 genes showing splicing differences among these species. RT–PCR analysis of 40 exons confirmed the predicted splicing evolution of 33 exons. Of these 33 exons, outgroup analysis using rhesus macaques confirmed 13 exons with human-specific increase or decrease in transcript inclusion levels after humans diverged from chimpanzees. Some of the human-specific brain splicing patterns disrupt domains critical for protein–protein interactions, and some modulate translational efficiency of their host genes. Strikingly, for exons showing splicing differences across species, we observed a significant increase in the rate of silent substitutions within exons, coupled with accelerated sequence divergence in flanking introns. This indicates that evolution of cis-regulatory signals is a major contributor to the emergence of human-specific splicing patterns. In one gene (MAGOH), using minigene reporter assays, we demonstrated that the combination of two human-specific cis-sequence changes created its human-specific splicing pattern. Together, our data reveal widespread human-specific changes of alternative splicing in the brain and suggest an important role of splicing in the evolution of neuronal gene regulation and functions. PMID:20460271

  19. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-m