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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. 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.

  3. An allosteric self-splicing ribozyme triggered by a bacterial second messenger.

    PubMed

    Lee, Elaine R; Baker, Jenny L; Weinberg, Zasha; Sudarsan, Narasimhan; Breaker, Ronald R

    2010-08-13

    Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

  4. Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake.

    PubMed Central

    Bramlage, B; Alefelder, S; Marschall, P; Eckstein, F

    1999-01-01

    Two synthetic hammerhead ribozymes, one unmodified and the other with 2"-modifications and four phosphorothioate groups, targeting a single GUA site in the luciferase mRNA, were compared for their inhibition of gene expression in cell cultureand their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was treated with these ribozymes by liposome-mediated transfection to determine their activity.Luciferase expression in cells was inhibited to approximately 50% with little difference between the unmodified and the 2"-modified ribozyme. A similar degree of inhibition was observed with two catalytically inactive ribozymes, indicating that inhibition was mainly due to an antisense effect. A ribozyme carrying a cholesterol moiety, applied to the cells without carrier, showed no inhibition. Northern blotting indicated a similar amount of cellular uptake of all ribozymes. The unmodified ribozyme was essentially evenly distributed between cytoplasm and nucleus, whereas a higher proportion of the phosphorothioate-containing ribozyme was observed in the nucleus. Fluorescence microscopy, including confocal microscopy using 5"-fluorescein-labelled ribozymes, showed that the unmodified and 2"-modified ribozymes were present in the cytoplasm and in the nucleus to a similar extent, whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expression to a comparable degree, suggesting that the ribozyme in the nucleus did not contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their inability to interfere with gene expression. PMID:10454613

  5. 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

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

    PubMed Central

    Monachello, Dario; Michel, François; Costa, Maria

    2016-01-01

    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. 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.

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

    PubMed

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

    2012-11-01

    Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. 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 circuit. 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.

  9. 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

  10. Hammerhead ribozyme cleavage of hamster prion pre-mRNA in complex cell-free model systems.

    PubMed

    Denman, R B; Purow, B; Rubenstein, R; Miller, D L

    1992-07-31

    The cleavage properties of a trans-acting hammerhead ribozyme targeted 51 bases upstream of the putative splicing branch point in the hamster prion pre-mRNA intron were investigated in cell-free model systems in vitro. The specificity of cleavage was demonstrated by the inability of this ribozyme to cleave a non-homologous synthetic message encoding part of the beta amyloid peptide precursor, beta APP, and by the inability of the prion pre-mRNA to be cleaved by a ribozyme targeted to beta amyloid peptide precursor mRNA. Also, the addition of total RNA isolated from rat brain had only a minimal effect on the cleavage of the prion substrate pre-mRNA by the ribozyme. Finally neither the presence of 100 ng of nuclear or cytoplasmic proteins were found to affect the rate of cleavage in vitro.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Probing the Tetrahymena group I ribozyme reaction in both directions.

    PubMed

    Karbstein, Katrin; Carroll, Kate S; Herschlag, Daniel

    2002-09-17

    The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.

  16. 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

  17. Metal Ions: Supporting Actors in the Playbook of Small Ribozymes

    PubMed Central

    Johnson-Buck, Alexander E.; McDowell, Sarah E.; Walter, Nils G.

    2012-01-01

    Since the 1980s, several small RNA motifs capable of chemical catalysis have been discovered. These small ribozymes, composed of between approximately 40 and 200 nucleotides, have been found to play vital roles in the replication of subviral and viral pathogens, gene regulation in prokaryotes, and have recently been discovered in noncoding eukaryotic RNAs. All of the known natural small ribozymes – the hairpin, hammerhead, hepatitis delta virus, Varkud satellite, and glmS ribozymes – catalyze the same self-cleavage reaction as RNAse A, resulting in two products, one bearing a 2′–3′ cyclic phosphate and the other a 5′-hydroxyl group. Although originally thought to be obligate metalloenzymes like the group I and II self-splicing introns, the small ribozymes are now known to support catalysis in a wide variety of cations that appear to be only indirectly involved in catalysis. Nevertheless, under physiologic conditions, metal ions are essential for the proper folding and function of the small ribozymes, the most effective of these being magnesium. Metal ions contribute to catalysis in the small ribozymes primarily by stabilizing the catalytically active conformation, but in some cases also by activating RNA functional groups for catalysis, directly participating in catalytic acid-base chemistry, and perhaps by neutralizing the developing negative charge of the transition state. Although interactions between the small ribozymes and cations are relatively nonspecific, ribozyme activity is quite sensitive to the types and concentrations of metal ions present in solution, suggesting a close evolutionary relationship between cellular metal ion homeostasis and cation requirements of catalytic RNAs, and perhaps RNA in general. PMID:22010272

  18. 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

  19. 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.

  20. 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

  1. A natural ribozyme with 3′,5′ RNA ligase activity

    PubMed Central

    Vicens, Quentin; Cech, Thomas R

    2010-01-01

    Using electrophoresis, sequencing and enzymatic digestion, we show that the group I intron from the cyanobacterium Anabaena sp. PCC 7120 catalyzes phosphodiester bond formation using a triphosphate on the 5′-terminal nucleotide, much like protein polymerases and engineered ribozymes. In the process, this ribozyme forms a unique circular RNA that incorporates the exogenous guanosine cofactor added during self-splicing. This finding may have relevance to a prebiotic RNA world and to modern biology. PMID:19125157

  2. 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

  3. Deep Sequencing Analysis of Aptazyme Variants Based on a Pistol Ribozyme.

    PubMed

    Kobori, Shungo; Takahashi, Kei; Yokobayashi, Yohei

    2017-04-14

    Chemically regulated self-cleaving ribozymes, or aptazymes, are emerging as a promising class of genetic devices that allow dynamic control of gene expression in synthetic biology. However, further expansion of the limited repertoire of ribozymes and aptamers, and development of new strategies to couple the RNA elements to engineer functional aptazymes are highly desirable for synthetic biology applications. Here, we report aptazymes based on the recently identified self-cleaving pistol ribozyme class using a guanine aptamer as the molecular sensing element. Two aptazyme architectures were studied by constructing and assaying 17 728 mutants by deep sequencing. Although one of the architectures did not yield functional aptazymes, a novel aptazyme design in which the aptamer and the ribozyme were placed in tandem yielded a number of guanine-inhibited ribozymes. Detailed analysis of the extensive sequence-function data suggests a mechanism that involves a competition between two mutually exclusive RNA structures reminiscent of natural bacterial riboswitches.

  4. Group I Ribozymes as a Paradigm for RNA Folding and Evolution

    NASA Astrophysics Data System (ADS)

    Woodson, Sarah A.; Chauhan, Seema

    Group I ribozymes are an ancient class of RNA catalysts that serve as a paradigm for the self-assembly of complex structures of non-coding RNA. The diversity of subtypes illustrates the modular character of RNA architecture and the potential for the evolution of new functions. The folding mechanisms of group I ribozymes illustrate the hierarchy of folding transitions and the importance of kinetic partitioning among competing folding pathways. Studies on group I splicing factors demonstrate how proteins facilitate the assembly of splicing complexes by stabilizing tertiary interactions between domains and by ATP-dependent cycles of RNA unfolding.

  5. 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

  6. Use of an engineered ribozyme to produce a circular human exon.

    PubMed Central

    Mikheeva, S; Hakim-Zargar, M; Carlson, D; Jarrell, K

    1997-01-01

    We report the use of an engineered ribozyme to produce a circular human exon in vitro. Specifically, we have designed a derivative of a yeast self-splicing group II intron that is able to catalyze the formation of a circular exon encoding the first kringle domain (K1) of the human tissue plasminogen activator protein. We show that the circular K1 exon is formed with high fidelity in vitro. Furthermore, the system is designed such that the circular exon that is produced consists entirely of human exon sequence. Thus, our results demonstrate that all yeast exon sequences are dispensable for group II intron catalyzed inverse splicing. This is the first demonstration that an engineered ribozyme can be used to create a circular exon containing only human sequences, linked together at a precise desired ligation point. We expect these results to be generalizable, so that similar ribozymes can be designed to precisely create circular derivatives of any nucleotide sequence. PMID:9396820

  7. 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

  8. Design and function of triplex hairpin ribozymes.

    PubMed

    Aquino-Jarquin, Guillermo; Rojas-Hernández, Ramiro; Alvarez-Salas, Luis Marat

    2010-01-01

    Triplex ribozymes allow for the individual activity of multiple trans-acting ribozymes producing higher target cleavage relative to tandem-expressed RZs. A triplex expression system based on a single hairpin ribozyme for the multiple expression (multiplex) vectors can be engineered to target RNAs with single or multiple antisense-accessible sites. System construction relies on triplex expression modules consisting of hairpin ribozyme cassettes flanked by ribozymes lacking catalytic domains. Multiplex vectors can be generated with single or multiple specificity by tandem cloning of triplex expression modules. Triplex ribozymes are initially tested in vitro using cis- and trans-cleavage assays against radioactive-labeled targets. In addition, triplex ribozymes are tested for cis and trans cleavage in vivo by transfection in cultured cells followed by ribonuclease protection assays (RPAs) and RT-PCR. The use of triplex configurations with multiplex ribozymes will provide the basis for the development of future RZ-based therapies and technologies.

  9. An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme.

    PubMed

    Nielsen, Henrik; Westhof, Eric; Johansen, Steinar

    2005-09-02

    Twin-ribozyme introns are formed by two ribozymes belonging to the group I family and occur in some ribosomal RNA transcripts. The group I-like ribozyme, GIR1, liberates the 5' end of a homing endonuclease messenger RNA in the slime mold Didymium iridis. We demonstrate that this cleavage occurs by a transesterification reaction with the joining of the first and the third nucleotide of the messenger by a 2',5'-phosphodiester linkage. Thus, a group I-like ribozyme catalyzes an RNA branching reaction similar to the first step of splicing in group II introns and spliceosomal introns. The resulting short lariat, by forming a protective 5' cap, might have been useful in a primitive RNA world.

  10. Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

    PubMed

    Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

    2017-03-17

    The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

  11. 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

  12. Optimizing ribozymes for somatic cell gene therapy.

    PubMed

    Branch, A D; Klotman, P E

    1998-01-01

    Therapeutic ribozymes are created through a multistep process that requires trial and error. There are few established rules governing ribozyme design, but guidelines are emerging. It is not yet known whether hammerheads and hairpins, the two ribozymes most widely studied as potential gene therapy agents, have the inherent capability to ablate single genes. Their capacity for specificity and selectivity remains to be explored through rigorous experimentation. These experiments require a battery of control molecules, the characteristics of which are outlined here. Methods for completing the steps in the ribozyme development process, from the selection of a target gene to the quantitation of RNA levels, are also presented and discussed.

  13. Visualizing group II intron catalysis through the stages of splicing

    PubMed Central

    Marcia, Marco; Pyle, Anna Marie

    2012-01-01

    SUMMARY Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report fourteen crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing, in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, adopting a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome. PMID:23101623

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. HDV-like self-cleaving ribozymes

    PubMed Central

    Webb, Chiu-Ho T

    2011-01-01

    HDV ribozymes catalyze their own scission from the transcript during rolling circle replication of the hepatitis delta virus. In vitro selection of self-cleaving ribozymes from a human genomic library revealed an HDV-like ribozyme in the second intron of the human CPEB3 gene and recent results suggest that this RNA affects episodic memory performance. Bioinformatic searches based on the secondary structure of the HDV/CPEB3 fold yielded numerous functional ribozymes in a wide variety of organisms. Genomic mapping of these RNAs suggested several biological roles, one of which is the 5′ processing of non-LTR retrotransposons. The family of HDV-like ribozymes thus continues to grow in numbers and biological importance. PMID:21734469

  20. A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction.

    PubMed

    Tasiouka, K I; Burke, J M

    1994-06-24

    Here, we show that a single RNA molecule derived from a group-I intron can provide the catalytic activity, the substrate recognition domain and the attacking nucleophile in a reaction that mimics the exon ligation step of splicing. To accomplish this reaction, we have linked a 5' exon sequence to the 3' end of an attenuated form of the self-splicing Tetrahymena rRNA intron. The ribozyme (I-E1) attacks an oligoribonucleotide analog of the 3' splice site (I'-E2) to generate a product containing ligated exons (I-E1-E2) and a small intron fragment (I'). Two modified introns were constructed and tested for activity. A construct designed to interact with the 3' splice site through intermolecular P9.0 and P10 helices was found to be inactive due to failure to form a stable ribozyme-substrate complex. A second modified intron and substrate combination was engineered, in which the complex was further stabilized by an intermolecular P9.2 helix. In this case, stable complexes and reaction products were identified. The reaction efficiency was low compared to splicing of the unmodified intron-containing precursor, and will be optimized in future experiments. Following optimization, we believe that this system may be exploited to examine the functional consequences of a wide variety of 3' splice-site modifications, and may provide the basis for development of highly selective trans-acting ribozymes.

  1. 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.

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

    PubMed Central

    McNeil, Bonnie A.; Zimmerly, Steven

    2014-01-01

    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. PMID:24751650

  3. Preparation and identification of anti-transforming growth factor β1 U1 small nuclear RNA chimeric ribozyme in vitro

    PubMed Central

    Lin, Ju-Sheng; Song, Yu-Hu; Kong, Xin-Juan; Li, Bin; Liu, Nan-Zhi; Wu, Xiao-Li; Jin, You-Xin

    2003-01-01

    AIM: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)β1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro. METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. 32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE. RESULTS: Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 °C; at 37 °C, it was active, Km = 34.48 nmol/L, Kcat = 0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct. CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future. PMID:12632521

  4. Conditional control of suicide gene expression in tumor cells with theophylline-responsive ribozyme.

    PubMed

    Zhang, Y; Wang, J; Cheng, H; Sun, Y; Liu, M; Wu, Z; Pei, R

    2017-02-01

    Numerous synthetic RNA-based controls for integrating sensing switches with function devices have been demonstrated in a variety of organisms for gene regulation. Although potential advantages of RNA-based genetic control strategies have been shown in clinical applications, successfully extending these engineered systems into medical applications has seldom been reported. Here, a synthetic RNA-based ribozyme system and its application in advancing rationally designed cellular therapy were described. The theophylline-responsive, ribozyme-based device provided a powerful platform for suicide gene expression regulation in tumor cells. Moreover, we demonstrate the ability of our synthetic controller to modulate effectively the viability of the cells in response to drug input. Our RNA-based regulatory system could dose-dependently fine-tune transgene expression in mammalian cells and address urgent limitations in existing genetic control strategies for gene- and cell-based therapies in the future.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. A ribozyme that ligates RNA to protein.

    PubMed

    Baskerville, Scott; Bartel, David P

    2002-07-09

    We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5' terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzyme-nucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.

  10. Kinetic Analysis of δ Ribozyme Cleavage†

    PubMed Central

    Mercure, Stéphane; Lafontaine, Daniel; Ananvoranich, Sirinart; Perreault, Jean-Pierre

    2010-01-01

    The ability of δ ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (kcat/KM) was higher at 37 °C than at 56 °C. The cleavage reaction seems to be limited by the product release step at 37 °C and by the chemical cleavage step at 56 °C. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (kcat and KM) and dissociation constant (Kd) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2′-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the δ ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex. PMID:9836591

  11. A Self-Replicating Ligase Ribozyme

    NASA Technical Reports Server (NTRS)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  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 Central

    Kobori, Shungo

    2016-01-01

    Abstract 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. 2-C-methyluridine modified hammerhead ribozyme against the estrogen receptor.

    PubMed

    Pontiggia, Rodrigo; Pontiggia, Osvaldo; Simian, Marina; Montserrat, Javier M; Engels, Joachim W; Iribarren, Adolfo M

    2010-05-01

    A new synthesis of 2'-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5'-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor alpha protein (ER-alpha), and transfection experiments in MCF-7 cell line are also presented.

  16. Ribozyme modulation of lipopolysaccharide-induced tumor necrosis factor-alpha production by peritoneal cells in vitro and in vivo.

    PubMed

    Sioud, M

    1996-05-01

    We have utilized synthetic ribozymes to modulate the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) by peritoneal cells. Two hammerhead ribozymes (mRz1 and mRz2) were prepared by transcription in vitro and their activities in vitro and in vivo were investigated. Both ribozymes cleaved their RNA target with an apparent turnover number (kcat) of 2 min(-1), and inhibited TNF-alpha gene expression in vitro by 50% and 70%, respectively. When mRz1 and mRz2, entrapped in liposomes, were delivered into mice by intraperitoneal injection, they inhibited LPS-induced TNF-alpha gene expression in vivo with mRz2 being the most effective. This enhanced activity could result from the facilitation of catalysis by cellular endogenous proteins, since they specifically bind to mRz2 as compared to mRz1. Furthermore, a significant mRz2 activity can be recovered from peritoneal cells 2 days post-administration in vivo. The anti-TNF-alpha ribozyme treatment in vivo resulted in a more significant reduction of LPS-induced IFN-gamma protein secretion compared to IL-10. In contrast to this pleiotropic effect, the anti-TNF-alpha ribozyme treatment did not affect the heterogenous expression of Fas ligand by peritoneal cells, indicating the specificity of the treatment. Taken together, the present data indicate that the biological effects of TNF-alpha can be modulated by ribozymes. In addition, the data suggest that ribozymes can be administered in a drug-like manner, and therefore indicate their potential in clinical applications.

  17. Crystallographic analysis of small ribozymes and riboswitches.

    PubMed

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

    2012-01-01

    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 preQ(1) riboswitch, and variants of a larger class II preQ(1) 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.

  18. 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

  19. Schistosome satellite DNA encodes active hammerhead ribozymes.

    PubMed

    Ferbeyre, G; Smith, J M; Cedergren, R

    1998-07-01

    Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

  20. In vitro Selection of glmS Ribozymes

    PubMed Central

    Link, Kristian H.; Breaker, Ronald R.

    2017-01-01

    Summary Riboswitches modulate gene expression in eubacteria and eukaryotes in response to changing concentrations of small molecule metabolites. In most examples studied to date, riboswitches achieve both metabolite sensing and gene control functions without the obligate involvement of protein factors. These findings validate the hypothesis that RNA molecules could be engineered to function as designer gene control elements that sense and respond to different ligands. We believe that reverse engineering natural riboswitches could provide an intellectual foundation for those who wish to build synthetic riboswitches. Also, natural riboswitches might serve as starting points for efforts to change ligand specificity or gene control function through mutation and selection in vitro. In this chapter, we describe how in vitro selection can be used to create variant glmS ribozymes. Additionally, we discuss how these techniques can be extended to other riboswitch classes. PMID:19381572

  1. Application of computational technologies to ribozyme biotechnology products

    NASA Astrophysics Data System (ADS)

    Christoffersen, Ralph E.; McSwiggen, James; Konings, Danielle

    1994-07-01

    Ribozymes are RNA molecules that act enzymatically to cleave other RNA molecules. The cleavage reaction requires the binding of ribozyme to specific sites on the target RNA through (mostly) Watson-Crick base-pairing interactions. Association of ribozyme with target completes a three-dimensional ribozyme/target complex which results in cleavage of the target RNA. We are employing both computational and experimental approaches to identify sites on target RNA molecules that are open to ribozyme attack and to determine which ribozymes are most active against those sites. Two types of computational technologies are available for aiding in the identification of target sites and design of active ribozymes. First, DNA/RNA sequence analysis software is employed to identify sequence motifs necessary for ribozyme cleavage and to look for sequence conservation between different sources of the target organism so that ribozymes with the broadest possible target range can be designed. Second, RNA folding algorithms are employed to predict the secondary structure of both ribozyme and target RNA in an attempt to identify combinations of ribozyme and target site that will successfully associate prior to ribozyme cleavage. The RNA folding algorithms utilize a set of thermodynamic parameters obtained from measurements on short RNA duplexes; while these rules give reasonable predictions of secondary structure for a small set of highly structured RNAs, they remain largely untested for predicting the structure of messenger RNAs. This paper outlines the current status of designing ribozymes that fold correctly and of locating target sites that are sufficiently unfolded to allow ribozyme cleavage.

  2. Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns.

    PubMed

    Asakura, Yukari; Barkan, Alice

    2006-12-01

    Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.

  3. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

    PubMed Central

    Collis, S. J.; Tighe, A.; Scott, S. D.; Roberts, S. A.; Hendry, J. H.; Margison, G. P.

    2001-01-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20–50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation. PMID:11266555

  4. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

    PubMed

    Collis, S J; Tighe, A; Scott, S D; Roberts, S A; Hendry, J H; Margison, G P

    2001-04-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

  5. One Sequence, Two Ribozymes: Implications for the Emergence of New Ribozyme Folds

    NASA Astrophysics Data System (ADS)

    Schultes, Erik A.; Bartel, David P.

    2000-07-01

    We describe a single RNA sequence that can assume either of two ribozyme folds and catalyze the two respective reactions. The two ribozyme folds share no evolutionary history and are completely different, with no base pairs (and probably no hydrogen bonds) in common. Minor variants of this sequence are highly active for one or the other reaction, and can be accessed from prototype ribozymes through a series of neutral mutations. Thus, in the course of evolution, new RNA folds could arise from preexisting folds, without the need to carry inactive intermediate sequences. This raises the possibility that biological RNAs having no structural or functional similarity might share a common ancestry. Furthermore, functional and structural divergence might, in some cases, precede rather than follow gene duplication.

  6. 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

  7. Assembly and activation of a kinase ribozyme

    PubMed Central

    Burke, Donald H.; Rhee, Steven S.

    2010-01-01

    RNA activities can be regulated by modulating the relative energies of all conformations in a folding landscape; however, it is often unknown precisely how peripheral elements perturb the overall landscape in the absence of discrete alternative folds (inactive ensemble). This work explores the effects of sequence and secondary structure in governing kinase ribozyme activity. Kin.46 catalyzes thiophosphoryl transfer from ATPγS onto the 5′ hydroxyl of polynucleotide substrates, and is regulated 10,000-fold by annealing an effector oligonucleotide to form activator helix P4. Transfer kinetics for an extensive series of ribozyme variants identified several dispensable internal single-stranded segments, in addition to a potential pseudoknot at the active site between segments J1/4 and J3/2 that is partially supported by compensatory rescue. Standard allosteric mechanisms were ruled out, such as formation of discrete repressive structures or docking P4 into the rest of the ribozyme via backbone 2′ hydroxyls. Instead, P4 serves both to complete an important structural element (100-fold contribution to the reaction relative to a P4-deleted variant) and to mitigate nonspecific, inhibitory effects of the single-stranded tail (an additional 100-fold contribution to the apparent rate constant, kobs). Thermodynamic activation parameters ΔH‡ and ΔS‡, calculated from the temperature dependence of kobs, varied with tail length and sequence. Inhibitory effects of the unpaired tail are largely enthalpic for short tails and are both enthalpic and entropic for longer tails. These results refine the structural view of this kinase ribozyme and highlight the importance of nonspecific ensemble effects in conformational regulation by peripheral elements. PMID:20935068

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. A conformational switch controls hepatitis delta virus ribozyme catalysis.

    PubMed

    Ke, Ailong; Zhou, Kaihong; Ding, Fang; Cate, Jamie H D; Doudna, Jennifer A

    2004-05-13

    Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.

  14. 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

  15. 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

  16. 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

  17. Inhibition of HIV-1 gp41 expression with hammerhead ribozymes.

    PubMed

    Fedoruk-Wyszomirska, Agnieszka; Szymański, Maciej; Głodowicz, Paweł; Gabryelska, Marta; Wyszko, Eliza; Estrin, William J; Barciszewski, Jan

    2015-10-01

    Despite great progress in the treatment of AIDS, HIV-1 remains one of the major concerns as a human pathogen. One of the therapeutic strategies against viral infections is the application of catalytic ribonucleic acids (ribozymes) that can significantly reduce expression of a target gene by site-specific hydrolysis of its mRNA. In the present paper, we report a study on the activity of several variants of hammerhead ribozymes targeting a conserved region within mRNA encoding HIV-1 envelope glycoprotein gp41. On the basis of the data from in vitro assays and gene silencing in the cultured cells, we propose a new hammerhead ribozyme targeting the gp41-encoding sequence that can be potentially used as a therapeutic agent in AIDS treatment. Moreover, we demonstrate that the hydrolytic activity of the ribozyme in the intracellular environment cannot be inferred solely from the results of in vitro experiments.

  18. 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

  19. Chemistry and biology of self-cleaving ribozymes

    PubMed Central

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

    2015-01-01

    Self-cleaving ribozymes were discovered thirty 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 employed 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

  20. Structural roles of monovalent cations in the HDV ribozyme.

    PubMed

    Ke, Ailong; Ding, Fang; Batchelor, Joseph D; Doudna, Jennifer A

    2007-03-01

    The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. Ribozyme genes protecting transgenic melon plants against potyviruses.

    PubMed

    Huttner, E; Tucker, W; Vermeulen, A; Ignart, F; Sawyer, B; Birch, R

    2001-04-01

    Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable). Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants. The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance (see for example 14). In the light of concerns expressed by some plant virologists (13) about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.

  10. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

    PubMed Central

    Hendry, P; McCall, M J; Santiago, F S; Jennings, P A

    1992-01-01

    Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate. PMID:1280808

  11. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

    PubMed Central

    Potratz, Jeffrey P.; Campo, Mark Del; Wolf, Rachel Z.; Lambowitz, Alan M.; Russell, Rick

    2011-01-01

    The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize non-native intronic structure. Nevertheless, Mss116p acceleration of all three constructs depends upon ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mtDNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing conversion of non-functional to functional RNA structure. PMID:21679717

  12. 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.

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

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-03

    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+.

  14. 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

  15. Splice-switching antisense oligonucleotides as therapeutic drugs

    PubMed Central

    Havens, Mallory A.; Hastings, Michelle L.

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  16. 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

  17. 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

  18. 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

  19. Antibiotic inhibition of group I ribozyme function.

    PubMed

    von Ahsen, U; Davies, J; Schroeder, R

    1991-09-26

    The discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have coevolved in the formation of the modern ribosome. Here we report that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli, inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide and an amino acid, but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.

  20. Kinetic partitioning mechanism of HDV ribozyme folding.

    PubMed

    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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Splicing plastic optical fibers

    NASA Astrophysics Data System (ADS)

    Carson, Susan D.; Salazar, Roberto A.

    1991-12-01

    Polymethylmethacrylate (PMMA) plastic optical fiber (500 micrometers diameter, fluoropolymer cladding) has been spliced using a fused silica sleeve and a variety of solvent/PMMA solutions as adhesives. Mechanical splicing using index matching fluid has also been investigated. To ensure good bonding and minimize scattering, fiber ends are polished prior to application of adhesive. Using an LED ((lambda) max approximately 640 nm), losses are routinely less than 1.0 dB/splice, and some adhesive formulations have exhibited losses as low as 0.2 dB/splice. Five-meter fibers with as many as ten splices/fiber have been monitored over a period of several months. No fiber has exhibited an increase in optical loss with time.

  6. Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA.

    PubMed

    Paukstelis, Paul J; Chen, Jui-Hui; Chase, Elaine; Lambowitz, Alan M; Golden, Barbara L

    2008-01-03

    The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.

  7. 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

  8. 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

  9. 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.

  10. Amplification of RNA by an RNA polymerase ribozyme

    PubMed Central

    Horning, David P.; Joyce, Gerald F.

    2016-01-01

    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

  11. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  12. In vitro selection of adenine-dependent ribozyme against Tpl2/Cot oncogene.

    PubMed

    Li, Yan-Li; Vergne, Jacques; Torchet, Claire; Maurel, Marie-Christine

    2009-01-01

    Hairpin ribozymes possess the properties of RNA sequence-specific recognition and site-specific cleavage. These properties make them a powerful extension of the antisense approach for the inhibition of gene expression. From a randomized RNA pool of hairpin ribozymes, using the systematic evolution of ligands by exponential enrichment, we have obtained an adenine-dependent hairpin ribozyme, Tpl2/Cot (tumour progression locus 2) ribozyme, which cleaves the Tpl2/Cot kinase mRNA sequence at nucleotides A225/G226 relative to the start codon of translation. This serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in cancer. The selected 'Tpl2/Cot-YL ribozyme' efficiently cleaves its target sequence in cis and in trans; furthermore, the ribozyme efficiently cleaves a longer target sequence of 54 nucleotides in trans, as well as the full-length mRNA.

  13. RNA helicases in splicing.

    PubMed

    Cordin, Olivier; Beggs, Jean D

    2013-01-01

    In eukaryotic cells, introns are spliced from pre-mRNAs by the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. There is evidence that the various helicases are functionally and physically connected with each other and with many other factors in the spliceosome. Understanding the dynamics of those interactions is essential to comprehend the mechanism and regulation of normal as well as of pathological splicing. This review focuses on recent advances in the characterization of the splicing helicases and their interactions, and highlights the deep integration of splicing helicases in global mRNP biogenesis pathways.

  14. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5′ splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5′ exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  15. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  16. Ribozyme Targeting the Novel Fusion Junction of EGFRvIII in Breast Cancer

    DTIC Science & Technology

    2003-07-01

    targeting the novel junction of EGFRvyII. * Demonstrate the therapeutic efficacy of an anti-EGFRvIll hammerhead ribozyme targeting the endogenous...first demonstration of the therapeutic efficacy of an anti-EGFRvlII hammerhead ribozyme targeting the endogenous EGFRvAII expression against human...202-687-7505.designed and generated a tumor specific hammerhead ribozyme E-mail: Tangc@georgetown.edu targeted to the novel fusion junction of

  17. Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2'-O-methylation.

    PubMed Central

    Goodchild, J

    1992-01-01

    RNA catalysts (ribozymes) designed to cleave sequences unique to viral RNA's might be developed as therapeutics. For this purpose, they would require high catalytic efficiency and resistance to nucleases. Reported here are two approaches that can be used in combination to improve these properties. First, catalytic efficiency can be improved by oligonucleotides (facilitators) that bind to the substrate contiguously with the 3'-end of the ribozyme. Second, 2'-O-methylation of flanking sequences of the ribozyme increases catalytic activity as well as resistance to nucleases. In combination with a facilitator oligodeoxynucleotide, the cleavage rate was increased 20 fold over that of the unmodified ribozyme. Images PMID:1383929

  18. 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.

  19. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  20. Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation.

    PubMed

    Gaines, Colin S; York, Darrin M

    2016-03-09

    We present results from molecular dynamics simulations and free energy calculations of the twister ribozyme at different stages along the reaction path to gain insight into its mechanism. The results, together with recent biochemical experiments, provide support for a mechanism involving general-acid catalysis by a conserved adenine residue in the active site. Although adenine has been previously implicated as a general acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be a twist. Biochemical experiments suggest that general acid catalysis may occur through the N3 position, which has never before been implicated in this role; however, currently, there is a lack of a detailed structural model for the active state of the twister ribozyme in solution that is consistent with these and other experiments. Simulations in a crystalline environment reported here are consistent with X-ray crystallographic data, and suggest that crystal packing contacts trap the RNA in an inactive conformation with U-1 in an extruded state that is incompatible with an in-line attack to the scissile phosphate. Simulations in solution, on the other hand, reveal this region to be dynamic and able to adopt a conformation where U-1 is stacked with G33. In this state, the nucleophile is in line with the scissile phosphate, and the N1 position of G33 and N3 position of A1 are poised to act as a general base and acid, respectively, as supported by mutational experiments. Free energy calculations further predict the electrostatic environment causes a shift of the microscopic pKa at the N3 position of A1 toward neutrality by approximately 5 pKa units. These results offer a unified interpretation of a broad range of currently available experimental data that points to a novel mode of general acid catalysis through the N3 position of an adenine nucleobase, thus expanding the repertoire of known mechanistic strategies employed by

  1. Ribozyme catalysis with a twist: the active state of the twister ribozyme in solution predicted from molecular simulation

    PubMed Central

    Gaines, Colin S.; York, Darrin M.

    2016-01-01

    We present results from molecular dynamics simulations and free energy calculations of the twister ribozyme at different stages along the reaction path in order to gain insight into its mechanism. Results, together with recent biochemical experiments, provide support for a mechanism involving general acid catalysis by a conserved adenine residue in the active site. Although adenine has been previously implicated as a general acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be - a twist. Biochemical experiments suggest that general acid catalysis may occur through the N3 position, which has never before been implicated in this role; however, there currently lacks a detailed structural model for the active state of the twister ribozyme in solution that is consistent with these and other experiments. Simulations in a crystalline environment reported here are consistent with X-ray crystallographic data, and suggest that crystal packing contacts trap the RNA in an inactive conformation with U-1 in an extruded state that is incompatible with an in-line attack to the scissile phosphate. Simulations in solution, on the other hand, reveal this region to be dynamic and able to adopt a conformation where U-1 is stacked with G33. In this state, the nucleophile is in line with the scissile phosphate, and the N1 position of G33 and N3 position of A1 are poised to act as general base and acid, respectively, as supported by mutational experiments. Free energy calculations further predict the electrostatic environment causes a shift of the microscopic pKa at the N3 position of A1 toward neutrality by approximately 5 pKa units. These results offer a unified interpretation of a broad range of currently available experimental data that points to a novel mode of general acid catalysis through the N3 position of an adenine nucleobase, thus expanding the repertoire of known mechanistic strategies employed by

  2. Hairpin ribozyme-catalyzed ligation in water-alcohol solutions.

    PubMed

    Vlassov, Alexander V; Johnston, Brian H; Kazakov, Sergei A

    2005-12-01

    The hairpin ribozyme (HPR) is a naturally existing RNA that catalyzes site-specific RNA cleavage and ligation. At 37 degrees C and in the presence of divalent metal ions (M(2+)), the HPR efficiently cleaves RNA substrates in trans. Here, we show that the HPR can catalyze efficient M(2+)-independent ligation in trans in aqueous solutions containing any of several alcohols, including methanol, ethanol, and isopropanol, and millimolar concentrations of monovalent cations. Ligation proceeds most efficiently in 60% isopropanol at 37 degrees C, whereas the reverse (cleavage) reaction is negligible under these conditions. We suggest that dehydration of the RNA is the key factor promoting HPR activity in water- alcohol solutions. Alcohol-induced ribozyme ligation may have practical applications.

  3. 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

  4. 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

  5. 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

  6. A triplex ribozyme expression system based on a single hairpin ribozyme.

    PubMed

    Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

    2008-09-01

    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 transcleavage 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.

  7. 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

  8. Investigating a New Generation of Ribozymes in Order to Target HCV

    PubMed Central

    Lévesque, Michel V.; Lévesque, Dominique; Brière, Francis P.; Perreault, Jean-Pierre

    2010-01-01

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

  9. Alternative splicing interference by xenobiotics.

    PubMed

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

  10. Liposome membrane can induce self-cleavage of RNA that models the core fragments of hammerhead ribozyme.

    PubMed

    Suga, Keishi; Tanaka, Seishiro; Umakoshi, Hiroshi

    2016-01-01

    The hammerhead ribozyme (HHR) is one of smallest catalytic RNAs, composed of a catalytic core and three stems; it undergoes self-cleavage in the presence of divalent magnesium ions (Mg(2+)) or other cations. It is hypothesized that the function and metabolism of RNAs might be regulated via interaction with lipid membranes in the prebiotic world. Using synthetic RNAs that model the core fragment of hammerhead ribozyme-like assembly (HHR-a), we investigated the enhancement of the self-cleavage reaction of HHR-a induced by the liposomes, both in the absence and presence of Mg(2+). The HHR-a activity was enhanced by 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC) = 8/2 liposome with Mg(2+), while other liposomes did not so significant. In the presence of DOPE/DPPC = 8/2 liposome, the HHR-a activity was observed without Mg(2+), revealed by the conformational change of the HHR inhibitor complex induced by the interaction with the liposome. The UV resonance Raman spectroscopy analysis investigated the interaction between lipid molecules and nucleobases, suggesting that the ethanolamine group of DOPE molecules are assumed to act as monovalent cations alternative to Mg(2+), depending on the liposome membrane characteristics.

  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. Prebiotic Factors Influencing the Activity of a Ligase Ribozyme.

    PubMed

    Anella, Fabrizio; Danelon, Christophe

    2017-04-06

    An RNA-lipid origin of life scenario provides a plausible route for compartmentalized replication of an informational polymer and subsequent division of the container. However, a full narrative to form such RNA protocells implies that catalytic RNA molecules, called ribozymes, can operate in the presence of self-assembled vesicles composed of prebiotically relevant constituents, such as fatty acids. Hereby, we subjected a newly engineered truncated variant of the L1 ligase ribozyme, named tL1, to various environmental conditions that may have prevailed on the early Earth with the objective to find a set of control parameters enabling both tL1-catalyzed ligation and formation of stable myristoleic acid (MA) vesicles. The separate and concurrent effects of temperature, concentrations of Mg(2+), MA, polyethylene glycol and various solutes were investigated. The most favorable condition tested consists of 100 mM NaCl, 1 mM Mg(2+), 5 mM MA, and 4 °C temperature, whereas the addition of Mg(2+)-chelating solutes, such as citrate, tRNAs, aspartic acid, and nucleoside triphosphates severely inhibits the reaction. These results further solidify the RNA-lipid world hypothesis and stress the importance of using a systems chemistry approach whereby a wide range of prebiotic factors interfacing with ribozymes are considered.

  13. 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

  14. 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

  15. Structure of ribonuclease P--a universal ribozyme.

    PubMed

    Torres-Larios, Alfredo; Swinger, Kerren K; Pan, Tao; Mondragón, Alfonso

    2006-06-01

    Ribonuclease P (RNase P) is one of only two known universal ribozymes and was one of the first ribozymes to be discovered. It is involved in RNA processing, in particular the 5' maturation of tRNA. Unlike most other natural ribozymes, it recognizes and cleaves its substrate in trans. RNase P is a ribonucleoprotein complex containing one RNA subunit and as few as one protein subunit. It has been shown that, in bacteria and in some archaea, the RNA subunit alone can support catalysis. The structure and function of bacterial RNase P RNA have been studied extensively, but the detailed catalytic mechanism is not yet fully understood. Recently, structures of one of the structural domains and of the entire RNA component of RNase P from two different bacteria have been described. These structures provide the first atomic-level information on the structural assembly of the RNA component, and the regions involved in substrate recognition and catalysis. Comparison of these structures reveals a highly conserved core that comprises two universally conserved structural modules. Interestingly, the same structural core can be found in the context of different scaffolds.

  16. 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

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

    PubMed Central

    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

  19. Inhibition of Plasmodium falciparum ispH (lytB) gene expression by hammerhead ribozyme.

    PubMed

    Vinayak, Sumiti; Sharma, Yagya D

    2007-01-01

    The nonmevalonate pathway of isoprenoid biosynthesis in the apicoplast of the human malaria parasite Plasmodium falciparum is distinct from the mevalonate-dependent pathway of humans and thus a good drug target. We describe here the hammerhead ribozyme based cleavage of the ispH (lytB) gene transcript involved in the last step of this nonmevalonate pathway. Using RNA folding program, three hammerhead ribozymes named as RZ(876), RZ(1260), and RZ(1331) were predicted against ispH (lytB) mRNA. Messenger walk screening (RNaseH) assay confirmed the target accessibility for these ribozymes. All three ribozymes cleaved the target RNA in vitro but RZ(876) exhibited the highest catalytic potential (62.92%). Therefore, RZ(876) was chemically synthesized with appropriate chemical modifications to protect it from nuclease attack while using it for in vitro parasite growth inhibition assay. This ribozyme RZ(876) was able to inhibit 87.36% parasite growth at 30 microM concentration compared to the untreated culture. However, an absolute inhibition of 29.41% was achieved compared to the control ribozyme (RZ(ctrl)). Nonetheless, the growth inhibition effect was found to be sequence-specific as indicated by the decreased level of ispH (lytB) transcript after ribozyme treatment. In conclusion, we have identified the ispH (lytB) as a potential target whose transcript can be cleaved by a ribozyme RZ(876).

  20. 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.

  1. 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

  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. Experimental cancer gene therapy by multiple anti-survivin hammerhead ribozymes.

    PubMed

    Fei, Qi; Zhang, Hongyu; Fu, Lili; Dai, Xinlan; Gao, Baomei; Ni, Min; Ge, Chao; Li, Jinjun; Ding, Xia; Ke, Yuwen; Yao, Xuebiao; Zhu, Jingde

    2008-06-01

    To improve the efficacy of gene therapy for cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting the exposed regions of survivin mRNA. In addition to the in vitro characterization, which included a determination of the sequence specificity of cleavage by primer extension, assays for cell proliferation and for in vivo tumor growth were used to score for ribozyme efficiency. The resulting suppression of survivin expression induced mitotic catastrophe and cell death via the caspase-3-dependent pathway. Importantly, administration of the ribozyme adenoviruses inhibited tumor growth in a hepatocellular carcinoma xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically suppressed survivin and, as this combination targets all major forms of the survivin transcripts, produced the most potent anti-cancer effects. The adenoviruses carrying the multiple hammerhead ribozymes described in this report offered a robust gene therapy strategy against cancer.

  4. 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.

  5. 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

  6. RiboaptDB: A Comprehensive Database of Ribozymes and Aptamers

    PubMed Central

    Thodima, Venkata; Pirooznia, Mehdi; Deng, Youping

    2006-01-01

    Background Catalytic RNA molecules are called ribozymes. The aptamers are DNA or RNA molecules that have been selected from vast populations of random sequences, through a combinatorial approach known as SELEX. The selected oligo-nucleotide sequences (~200 bp in length) have the ability to recognize a broad range of specific ligands by forming binding pockets. These novel aptamer sequences can bind to nucleic acids, proteins or small organic and inorganic chemical compounds and have many potential uses in medicine and technology. Results The comprehensive sequence information on aptamers and ribozymes that have been generated by in vitro selection methods are included in this RiboaptDB database. Such types of unnatural data generated by in vitro methods are not available in the public 'natural' sequence databases such as GenBank and EMBL. The amount of sequence data generated by in vitro selection experiments has been accumulating exponentially. There are 370 artificial ribozyme sequences and 3842 aptamer sequences in the total 4212 sequences from 423 citations in this RiboaptDB. We included general search feature, and individual feature wise search, user submission form for new data through online and also local BLAST search. Conclusion This database, besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility in medicine, provides valuable information for computational and theoretical biologists. The RiboaptDB is extremely useful for garnering information about in vitro selection experiments as a whole and for better understanding the distribution of functional nucleic acids in sequence space. The database is updated regularly and is publicly available at . PMID:17118149

  7. 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

  8. RNA self-ligation: from oligonucleotides to full length ribozymes.

    PubMed

    Gwiazda, Slawomir; Salomon, Kristina; Appel, Bettina; Müller, Sabine

    2012-07-01

    The RNA-world-theory is one possible explanation of how life on earth has evolved. In this context it is of high interest to search for molecular systems, capable of self-organization into structures with increasing complexity. We have engineered a simple catalytic system in which two short RNA molecules can catalyze their own ligation to form a larger RNA construct. The system is based on the hairpin ribozyme using a 2',3'-cyclophosphate as activated species for ligation. 2',3'-cyclic phosphates can be easily formed and occur in many natural systems, thus being superior candidates for activated building blocks in RNA world scenarios.

  9. 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.

  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. Substrate specificity and reaction kinetics of an X-motif ribozyme

    PubMed Central

    LAZAREV, DENIS; PUSKARZ, IZABELA; BREAKER, RONALD R.

    2003-01-01

    The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions. PMID:12756327

  12. 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.

  13. Transcription and splicing: when the twain meet.

    PubMed

    Brody, Yehuda; Shav-Tal, Yaron

    2011-01-01

    Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

  14. Rolling Circle Transcription of Ribozymes Targeted to ras and mdr-1

    DTIC Science & Technology

    2001-09-01

    ssDNA) to direct transcription of an tion-PCR, and recyclization were carried out to optimize active hammerhead ribozyme in E. coli cells. transcription...transcription I hammerhead ribozyme I in vitro selection and 12.5 units/ml RNase inhibitor (Promega), in a total reaction volume of 15 tk1. After a...sequence encoding a ssDNA, and splint ssDNA were ethanol-precipitated and used as hammerhead ribozyme . templates to begin the next round of in vitro

  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 the active site of a diels-alderase ribozyme by photoaffinity cross-linking.

    PubMed

    Wombacher, Richard; Jäschke, Andres

    2008-07-09

    The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

  17. RNA splicing: disease and therapy.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2011-05-01

    The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.

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

    PubMed

    Przybilski, Rita; Hammann, Christian

    2007-10-01

    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.

  19. 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.

  20. 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

  1. 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

  2. Alternative RNA splicing and cancer.

    PubMed

    Liu, Sali; Cheng, Chonghui

    2013-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells.

  3. Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.

    PubMed Central

    Allain, F H; Varani, G

    1995-01-01

    The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition. Images PMID:7885828

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation.

    PubMed

    Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S; Naash, Muna I

    2016-01-01

    The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.

  10. Pressure modulates the self-cleavage step of the hairpin ribozyme.

    PubMed

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-03-30

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

  11. 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.

  12. Pressure modulates the self-cleavage step of the hairpin ribozyme

    PubMed Central

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-01-01

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties. PMID:28358002

  13. Long-distance communication in the HDV ribozyme: insights from molecular dynamics and experiments.

    PubMed

    Veeraraghavan, Narayanan; Bevilacqua, Philip C; Hammes-Schiffer, Sharon

    2010-09-10

    The hepatitis delta virus ribozyme is a small, self-cleaving RNA with a compact tertiary structure and buried active site that is important in the life cycle of the virus. The ribozyme's function in nature is to cleave an internal phosphodiester bond and linearize concatemers during rolling circle replication. Crystal structures of the ribozyme have been solved in both pre-cleaved and post-cleaved (product) forms and reveal an intricate network of interactions that conspire to catalyze bond cleavage. In addition, extensive biochemical studies have been performed to work out a mechanism for bond cleavage in which C75 and a magnesium ion catalyze the reaction by general acid-base chemistry. One issue that has remained unclear in this ribozyme and in other ribozymes is the nature of long-distance communication between peripheral regions of the RNA and the buried active site. We performed molecular dynamics simulations on the hepatitis delta virus ribozyme in the product form and assessed communication between a distal structural portion of the ribozyme-the protonated C41 base triple-and the active site containing the critical C75. We varied the ionization state of C41 in both the wild type and a C41 double mutant variant and determined the impact on the active site. In all four cases, effects at the active site observed in the simulations agree with experimental studies on ribozyme activity. Overall, these studies indicate that small functional RNAs have the potential to communicate interactions over long distances and that wild-type RNAs may have evolved ways to prevent such interactions from interfering with catalysis.

  14. 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

  15. 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

  16. 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.

  17. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

  18. cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs.

    PubMed

    Meyer, Mélanie; Masquida, Benoît

    2014-01-01

    RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.

  19. 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

  20. 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

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

    DOE PAGES

    Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; ...

    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

  2. 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

  3. 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.

  4. Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint.

    PubMed

    Gabryelska, Marta Magdalena; Wyszko, Eliza; Szymański, Maciej; Popenda, Mariusz; Barciszewski, Jan

    2013-05-01

    Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Owing to its small size and high activity, it is used as a model for RNA structure-function relationship studies. In the present paper we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells, but shows low activity in vitro. To understand it, we analysed tertiary structure models of substrate-ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.

  5. Cellular stress and RNA splicing.

    PubMed

    Biamonti, Giuseppe; Caceres, Javier F

    2009-03-01

    In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

  6. Splicing of aged fibers

    NASA Astrophysics Data System (ADS)

    Volotinen, Tarja T.; Yuce, Hakan H.; Bonanno, Nicholas; Frantz, Rolf A.; Duffy, Sean

    1993-11-01

    The deployment of fiber in the subscriber loop will require that an optical fiber network maintain the highest possible level of reliability over time, despite being subjected to extremes of temperature, humidity, and other environmental and mechanical stresses imposed on the outside plant. At the same time, both the initial cost and the ongoing maintenance expenses for loop equipment must be kept low. Fiber in the Loop (FITL) applications will entail increased fiber handling. Cable lengths will be shorter, and fiber counts higher, than has been the case so far in long-distance applications. There will also be more cable sheath openings per unit length of cable and/or fiber, as well as more splicing and connectorization. It may become a common practice that a customer is connected to a cable installed many years earlier. In subscriber loops, cables and fibers will be installed in harsher and more varying environments. Fibers will be exposed to higher humidity and temperature, particularly in splice boxes mounted on building walls, in pedestal cabinets, and in other similar enclosures. Corrosive gases and/or liquids may also be present at some locations and will adversely affect the fibers. The combination of increased handling, greater exposure, and more stressful environments may give rise to a need for new, more stringent requirements for fiber mechanical reliability. These can include increaSed fiber strength, increased aging resistance, and increased fatigue resistance.

  7. 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.

  8. A hammerhead ribozyme inhibits ADE1 gene expression in yeast.

    PubMed

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1995-03-21

    To study factors that affect in vivo ribozyme (Rz) activity, a model system has been devised in Saccharomyces cerevisiae based on the inhibition of ADE1 gene expression. This gene was chosen because Rz action can be evaluated visually by the Red phenotype produced when the activity of the gene product is inhibited. Different plasmid constructs allowed the expression of the Rz either in cis or in trans with respect to ADE1. Rz-related inhibition of ADE1 expression was correlated with a Red phenotype and a diminution of ADE1 mRNA levels only when the Rz gene was linked 5' to ADE1. The presence of the expected 3' cleavage fragment was demonstrated using a technique combining RNA ligation and PCR. This yeast system and detection technique are suited to the investigation of general factors affecting Rz-catalyzed inhibition of gene expression under in vivo conditions.

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

    PubMed

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

    2014-07-01

    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.

  10. 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

  11. In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron.

    PubMed Central

    Guo, Feng; Cech, Thomas R

    2002-01-01

    In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo. PMID:12022231

  12. 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

  13. 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

  14. 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

  15. Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.

    PubMed

    Kawasaki, Hiroaki; Onuki, Reiko; Suyama, Eigo; Taira, Kazunari

    2002-04-01

    Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Crystal structure of Pistol, a class of self-cleaving ribozyme.

    PubMed

    Nguyen, Laura A; Wang, Jimin; Steitz, Thomas A

    2017-01-31

    Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

  2. 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

  3. 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

  4. 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

  5. Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast.

    PubMed

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1996-08-09

    A hammerhead ribozyme designed to cleave the yeast ADE1 mRNA has been expressed in yeast under the control of a galactose-inducible promoter. RNA prepared from the galactose-induced yeast cultures possesses an activity that cleaves ADE1 mRNA in vitro. However, in spite of high expression levels of the ribozyme, no cleavage activity could be demonstrated in vivo. On the other hand, when the yeast cells expressing hammerhead RNA were treated with the alpha-factor mating pheromone, the level of ADE1 mRNA was reduced by 50%. Similar reductions were observed when this strain was cultured in the presence of lithium acetate or in nitrogen-free medium. Moreover, control experiments in which disabled hammerhead genes were expressed showed no such reductions. Extension of the length of the flanking recognition arms of the ribozyme from a total of 10 to 16 or 24 nucleotides diminished the inhibitory effect of the ribozyme. These data suggest that ribozymes are able to cleave a trans-RNA target in yeast.

  6. RNA splicing and splicing regulator changes in prostate cancer pathology.

    PubMed

    Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

    2017-04-05

    Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

  7. Targeting RNA Splicing for Disease Therapy

    PubMed Central

    Havens, Mallory A.; Duelli, Dominik M.

    2013-01-01

    Splicing of pre-messenger RNA into mature messenger RNA is an essential step for 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. PMID:23512601

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis

    PubMed Central

    Martick, Monika; Scott, William G.

    2015-01-01

    SUMMARY Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 Å resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner. PMID:16859740

  14. 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

  15. Extraordinary rates of transition metal ion-mediated ribozyme catalysis

    PubMed Central

    Roychowdhury-Saha, Manami; Burke, Donald H.

    2006-01-01

    In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme “RzB” cleaves its substrate RNA with maximal measured k obs values of ∼3000 min−1 in 1 mM Mn2+ and ∼780 min−1 in 1 mM Mg2+ at 37°C (pH 7.4). Apparent pKa for the catalytic general base is ∼7.8–8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd2+, for which the general catalytic acid has a pKa of 7.3 ± 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be ∼9.5 in Mn2+ and >9.5 in Mg2+. The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by ∼1–2 pH units in the presence of Cd2+ and Mn2+. One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid. PMID:16912216

  16. Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

    PubMed Central

    Bonifert, Tobias; Gonzalez Menendez, Irene; Battke, Florian; Theurer, Yvonne; Synofzik, Matthis; Schöls, Ludger; Wissinger, Bernd

    2016-01-01

    Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2′O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (~55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (~16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ~3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs. PMID:27874857

  17. 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

  18. 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

  19. 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

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

    PubMed

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

    2015-10-20

    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 Zn(2+) 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.

  1. The Small Ribozymes: Common and Diverse Features Observed through the FRET Lens

    PubMed Central

    Walter, Nils G.; Perumal, Shiamalee

    2011-01-01

    The hammerhead, hairpin, HDV, VS and glmS ribozymes are the five known, naturally occurring catalytic RNAs classified as the “small ribozymes”. They share common reaction chemistry in cleaving their own backbone by phosphodiester transfer, but are diverse in their secondary and tertiary structures, indicating that Nature has found at least five independent solutions to a common chemical task. Fluorescence resonance energy transfer (FRET) has been extensively used to detect conformational changes in these ribozymes and dissect their reaction pathways. Common and diverse features are beginning to emerge that, by extension, highlight general biophysical properties of non-protein coding RNAs. PMID:21796234

  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. Promoter usage and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2005-06-01

    Recent findings justify a renewed interest in alternative splicing (AS): the process is more a rule than an exception as it affects the expression of 60% of human genes; it explains how a vast mammalian proteomic complexity is achieved with a limited number of genes; and mutations in AS regulatory sequences are a widespread source of human disease. AS regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to transcription. A clearer picture is emerging of the mechanisms by which transcription affects AS through promoter identity and occupation. These mechanisms involve the recruitment of factors with dual functions in transcription and splicing (i.e. that contain both functional domains and hence link the two processes) and the control of RNA polymerase II elongation.

  4. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  5. Exploring ribozyme conformational changes with X-ray crystallography.

    PubMed

    Spitale, Robert C; Wedekind, Joseph E

    2009-10-01

    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.

  6. Evolution of ribozymes in the presence of a mineral surface

    PubMed Central

    Stephenson, James D.; Popović, Milena; Bristow, Thomas F.

    2016-01-01

    Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes. These processes include both the initial emergence of functional structures in the presence of the mineral and the subsequent transition away from the mineral-associated niche. The initial evolution of function depends upon the number and distribution of sequences capable of functioning in the presence of the mineral, and the transition to new environments depends upon the overlap between sequences that evolve on the mineral surface and sequences that can perform the same functions in the mineral's absence. To examine these processes, we evolved self-cleaving ribozymes in vitro in the presence or absence of Na-saturated montmorillonite clay mineral particles. Starting from a shared population of random sequences, RNA populations were evolved in parallel, along separate evolutionary trajectories. Comparative sequence analysis and activity assays show that the impact of this clay mineral on functional structure selection was minimal; it neither prevented common structures from emerging, nor did it promote the emergence of new structures. This suggests that montmorillonite does not improve RNA's ability to evolve functional structures; however, it also suggests that RNAs that do evolve in contact with montmorillonite retain the same structures in mineral-free environments, potentially facilitating an evolutionary transition away from a mineral-associated niche. PMID:27793980

  7. Intracellular Immunization of Human Fetal Cord Blood Stem/Progenitor Cells with a Ribozyme Against Human Immunodeficiency Virus Type 1

    NASA Astrophysics Data System (ADS)

    Yu, Mang; Leavitt, Mark C.; Maruyama, Midori; Yamada, Osamu; Young, Dennis; Ho, Anthony D.; Wong-Staal, Flossie

    1995-01-01

    Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34^+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

  8. 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

  9. 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

  10. The structure of a nucleolytic ribozyme that employs a catalytic metal ion.

    PubMed

    Liu, Yijin; Wilson, Timothy J; Lilley, David M J

    2017-03-06

    The TS ribozyme (originally called "twister sister") is a catalytic RNA. We present a crystal structure of the ribozyme in a pre-reactive conformation. Two co-axial helical stacks are organized by a three-way junction and two tertiary contacts. Five divalent metal ions are directly coordinated to RNA ligands, making important contributions to the RNA architecture. The scissile phosphate lies in a quasihelical loop region that is organized by a network of hydrogen bonding. A divalent metal ion is directly bound to the nucleobase 5' to the scissile phosphate, with an inner-sphere water molecule positioned to interact with the O2' nucleophile. The rate of ribozyme cleavage correlated in a log-linear manner with divalent metal ion pKa, consistent with proton transfer in the transition state, and we propose that the bound metal ion is a likely general base for the cleavage reaction. Our data indicate that the TS ribozyme functions predominantly as a metalloenzyme.

  11. 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

  12. 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

  13. Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

    PubMed

    Lafontaine, D A; Ananvoranich, S; Perreault, J P

    1999-08-01

    We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.

  14. An efficient ligation reaction promoted by a Varkud Satellite ribozyme with extended 5'- and 3'-termini.

    PubMed

    Jones, F D; Ryder, S P; Strobel, S A

    2001-12-15

    The Neurospora Varkud Satellite (VS) RNA is capable of promoting a reversible self-cleavage reaction important for its replication pathway. In vivo the VS RNA performs a cis-cleavage reaction to generate monomeric length transcripts that are subsequently ligated to produce circular VS RNA. The predominant form of VS RNA observed in vivo is the closed circular form, though minimal VS ribozyme self-cleavage constructs lack detectable ligation activity. MFOLD analysis of the entire VS RNA sequence revealed an extended region 5' and 3' of the minimal self-cleaving region that could anneal to form a complementary helix, which we have termed helix 7. In full-length VS RNA, this helix appears to span over 40 bp of sequence and brings the 5'- and 3'-ends of the RNA into proximity for the ligation reaction. Here we report a variant of the VS ribozyme with an extended 5'- and 3'-terminus capable of forming a truncated helix 7 that promotes the ligation reaction in vitro. Through mutation and selection of this RNA we have identified a ribozyme containing two point mutations in the truncated helix 7 that ligates with >70% efficiency. These results show that an additional helical element absent in current VS ribozyme constructs is likely to be important for the ligation activity of VS RNA.

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

    PubMed

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

    2012-03-01

    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 (k(cat)/K(M)) 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.

  16. 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

  17. 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.

  18. Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

    PubMed

    Bingaman, Jamie L; Messina, Kyle J; Bevilacqua, Philip C

    2017-03-14

    Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions.

  19. 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

  20. 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.

  1. Ribozymes: catalytic RNAs that cut things, make things, and do odd and useful jobs.

    PubMed

    Walter, Nils G; Engelke, David R

    2002-10-01

    Catalytic RNAs, or ribozymes, are a fossil record of the ancient molecular evolution of life on earth and still provide the essential core of macromolecule synthesis in all life forms today. Are they also an avenue to the development of new catalysts to recreate evolution, or to use as therapeutics and molecule sensors.

  2. 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

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

    PubMed Central

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

    2016-01-01

    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 on the crystal structure of the env25 pistol ribozyme, 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 general base and general acid respectively to accelerate cleavage chemistry, with their roles confirmed from cleavage assays on mutants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defines how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on mutants identifying key residues participating in acid-base catalyzed cleavage chemistry. PMID:27398999

  4. 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

  5. NMR-spectroscopic characterization of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme.

    PubMed

    Fürtig, Boris; Richter, Christian; Schell, Peter; Wenter, Philipp; Pitsch, Stefan; Schwalbe, Harald

    2008-01-01

    In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.

  6. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    PubMed

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  7. The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

    PubMed

    Cottrell, Joseph W; Scott, Lincoln G; Fedor, Martha J

    2011-05-20

    Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

  8. 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

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

    PubMed

    Menger, M; Eckstein, F; Porschke, D

    2000-11-15

    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 approximately 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 (tau approximately 200 ms). Addition of Mg(2+) 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 Mg(2+) are very similar to those induced by Ca(2+). The relaxation data do not provide any evidence for formation of Mg(2+)-inner sphere complexes in hammerhead ribozymes, because a Mg(2+)-specific relaxation effect was not visible. However, a Mg(2+)-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2

  10. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  11. Evolutionary conservation of alternative splicing in chicken

    PubMed Central

    Katyal, S.; Gao, Z.; Liu, R.-Z.; Godbout, R.

    2013-01-01

    Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

  12. Alcoholism and alternative splicing of candidate genes.

    PubMed

    Sasabe, Toshikazu; Ishiura, Shoichi

    2010-04-01

    Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

  13. 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.

  14. Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme.

    PubMed

    Klostermeier, D; Millar, D P

    2000-10-24

    Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.

  15. 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

  16. Conserved RNA secondary structures promote alternative splicing.

    PubMed

    Shepard, Peter J; Hertel, Klemens J

    2008-08-01

    Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. Alternative splicing in higher eukaryotes results in the generation of multiple protein isoforms from gene transcripts. The extensive alternative splicing observed implies a flexibility of the spliceosome to identify exons within a given pre-mRNA. To reach this flexibility, splice-site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice-site strength, splicing regulators, the exon/intron architecture, and the process of pre-mRNA synthesis itself. RNA secondary structures have also been proposed to influence alternative splicing as stable RNA secondary structures that mask splice sites are expected to interfere with splice-site recognition. Using structural and functional conservation, we identified RNA structure elements within the human genome that associate with alternative splice-site selection. Their frequent involvement with alternative splicing demonstrates that RNA structure formation is an important mechanism regulating gene expression and disease.

  17. 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.

  18. 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

  19. Aberrant Alternative Splicing Is Another Hallmark of Cancer

    PubMed Central

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks. PMID:24101931

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants

    PubMed Central

    Bucher, Johan; Lammers, Michiel; Busscher-Lange, Jacqueline; Bonnema, Guusje; Rodenburg, Nicole; Proveniers, Marcel C. G.; Angenent, Gerco C.

    2017-01-01

    Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures. PMID:28257507

  9. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  10. Cryptic splice sites and split genes.

    PubMed

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

    2011-08-01

    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.

  11. 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

  12. Pharmacology of Modulators of Alternative Splicing

    PubMed Central

    Morris, Jonathan C.; Oltean, Sebastian; Donaldson, Lucy F.

    2017-01-01

    More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed. PMID:28034912

  13. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  14. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

  15. SYNTHETIC OIL,

    DTIC Science & Technology

    The patent concerns a dicarboxylate-base synthetic oil with antiwear and antioxidation additives. The oil is prepared from the esterification of 2- or 3-methylcyclohexanol and 2-ethylhexanol with adipic acid. (Author)

  16. 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

  17. 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.

  18. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.

  19. Activation of the glmS Ribozyme Confers Bacterial Growth Inhibition.

    PubMed

    Schüller, Anna; Matzner, Daniel; Lünse, Christina E; Wittmann, Valentin; Schumacher, Catherine; Unsleber, Sandra; Brötz-Oesterhelt, Heike; Mayer, Christoph; Bierbaum, Gabriele; Mayer, Günter

    2017-03-02

    The ever-growing number of pathogenic bacteria resistant to treatment with antibiotics call for the development of novel compounds with as-yet unexplored modes of action. Here, we demonstrate the in vivo antibacterial activity of carba-α-d-glucosamine (CGlcN). In this mode of action study, we provide evidence that CGlcN-mediated growth inhibition is due to glmS ribozyme activation, and we demonstrate that CGlcN hijacks an endogenous activation pathway, hence utilizing a prodrug mechanism. This is the first report describing antibacterial activity mediated by activating the self-cleaving properties of a ribozyme. Our results open the path towards a compound class with an entirely novel and distinct molecular mechanism.

  20. Identification of an imino group indispensable for cleavage by a small ribozyme.

    PubMed

    Spitale, Robert C; Volpini, Rosaria; Heller, Moriah G; Krucinska, Jolanta; Cristalli, Gloria; Wedekind, Joseph E

    2009-05-06

    The hairpin ribozyme is a small, noncoding RNA (ncRNA) that catalyzes a site-specific phosphodiester bond cleavage reaction. Prior biochemical and structural analyses pinpointed the amidine moiety of base Ade38 as a key functional group in catalysis, but base changes designed to probe function resulted in localized misfolding of the active site. To define the requirements for chemical activity using a conservative modification, we synthesized and incorporated N1-deazaadenosine into the full-length ribozyme construct. This single-atom variant severely impairs activity, although the active-site fold remains intact in the accompanying crystal structures. The results demonstrate the essentiality of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations. This work demonstrates the efficacy of single-atom approaches in the analysis of ncRNA structure-function relationships.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Rearrangement of substrate secondary structure facilitates binding to the Neurospora VS ribozyme.

    PubMed

    Zamel, Ricardo; Collins, Richard A

    2002-12-13

    The Neurospora VS ribozyme differs from other small, naturally occurring ribozymes in that it recognizes for trans cleavage or ligation a substrate that consists largely of a stem-loop structure. We have previously found that cleavage or ligation by the VS ribozyme requires substantial rearrangement of the secondary structure of stem-loop I, which contains the cleavage/ligation site. This rearrangement includes breaking the top base-pair of stem-loop I, allowing formation of a kissing interaction with loop V, and changing the partners of at least three other base-pairs within stem-loop I to adopt a conformation termed shifted. In the work presented, we have designed a binding assay and used mutational analysis to investigate the contribution of each of these structural changes to binding and ligation. We find that the loop I-V kissing interaction is necessary but not sufficient for binding and ligation. Constitutive opening of the top base-pair of stem-loop I has little, if any, effect on either activity. In contrast, the ability to adopt the shifted conformation of stem-loop I is a major determinant of binding: mutants that cannot adopt this conformation bind much more weakly than wild-type and mutants with a constitutively shifted stem-loop I bind much more strongly. These results implicate the adoption of the shifted structure of stem-loop I as an important process at the binding step in the VS ribozyme reaction pathway. Further investigation of features near the cleavage/ligation site revealed that sulphur substitution of the non-bridging phosphate oxygen atoms immediately downstream of the cleavage/ligation site, implicated in a putative metal ion binding site, significantly altered the cleavage/ligation equilibrium but did not perturb substrate binding significantly. This indicates that the substituted oxygen atoms, or an associated metal ion, affect a step that occurs after binding and that they influence the rates of cleavage and ligation differently.

  6. 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

  7. 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

  8. An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

    PubMed

    Szewczak, A A; Ortoleva-Donnelly, L; Zivarts, M V; Oyelere, A K; Kazantsev, A V; Strobel, S A

    1999-09-28

    Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

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

    PubMed

    Bouchard, Patricia; Legault, Pascale

    2014-09-01

    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.

  10. 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.

  11. [Deregulation of pre-messenger RNA splicing and rare diseases].

    PubMed

    de la Grange, Pierre

    2016-12-01

    Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein.

  12. Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury.

    PubMed

    Ando, Hideyuki; Fukuda, Noboru; Kotani, Motoko; Yokoyama, Shin ichiro; Kunimoto, Satoshi; Matsumoto, Koichi; Saito, Satoshi; Kanmatsuse, Katsuo; Mugishima, Hideo

    2004-01-12

    We designed and synthesized a chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor (TGF)-beta 1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-beta 1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-beta 1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-beta 1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-beta 1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-beta 1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty.

  13. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

    PubMed

    LaVail, M M; Yasumura, D; Matthes, M T; Drenser, K A; Flannery, J G; Lewin, A S; Hauswirth, W W

    2000-10-10

    Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

  14. Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme.

    PubMed

    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 U(23) and G(28) 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 U(23) and G(28) 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.

  15. A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

    PubMed

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

    2012-10-17

    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 pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) 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 pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports 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.

  16. 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

  17. 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.

  18. Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme.

    PubMed

    Xie, Zheng; Srividya, Narayanan; Sosnick, Tobin R; Pan, Tao; Scherer, Norbert F

    2004-01-13

    The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition. The present study focuses on the U-to-I(eq) transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition. Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds. The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches. A free energy contour is constructed to illustrate the complex folding surface.

  19. Magnesium-Dependent Active-Site Conformational Selection in the Diels-Alderase Ribozyme

    SciTech Connect

    Berezniak, Tomasz; Zahran, Mai; Imhof, Petra; Jaeschke, Andres; Smith, Jeremy C

    2010-10-01

    The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.

    PubMed

    Wu, J Y; Maniatis, T

    1993-12-17

    Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

  7. The incredible complexity of RNA splicing.

    PubMed

    Robert, Christelle; Watson, Mick

    2016-12-30

    Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets.

  8. Multiple links between transcription and splicing.

    PubMed

    Kornblihtt, Alberto R; de la Mata, Manuel; Fededa, Juan Pablo; Munoz, Manuel J; Nogues, Guadalupe

    2004-10-01

    Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

  9. 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

  10. 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.

  11. 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

  12. 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

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

    PubMed Central

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

    2016-01-01

    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

  14. 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.

  15. Tumor microenvironment-associated modifications of alternative splicing.

    PubMed

    Brosseau, Jean-Philippe; Lucier, Jean-François; Nwilati, Hanad; Thibault, Philippe; Garneau, Daniel; Gendron, Daniel; Durand, Mathieu; Couture, Sonia; Lapointe, Elvy; Prinos, Panagiotis; Klinck, Roscoe; Perreault, Jean-Pierre; Chabot, Benoit; Abou-Elela, Sherif

    2014-02-01

    Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

  16. Synthetic multicellularity.

    PubMed

    Maharbiz, Michel M

    2012-12-01

    The ability to synthesize biological constructs on the scale of the organisms we observe unaided is probably one of the more outlandish, yet recurring, dreams humans have had since they began to modify genes. This review brings together recent developments in synthetic biology, cell and developmental biology, computation, and technological development to provide context and direction for the engineering of rudimentary, autonomous multicellular ensembles.

  17. Synthetic DNA

    PubMed Central

    O’ Driscoll, Aisling; Sleator, Roy D.

    2013-01-01

    With world wide data predicted to exceed 40 trillion gigabytes by 2020, big data storage is a very real and escalating problem. Herein, we discuss the utility of synthetic DNA as a robust and eco-friendly archival data storage solution of the future. PMID:23514938

  18. 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.

  19. 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.

  20. 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

  1. An interspecific plant hybrid shows novel changes in parental splice forms of genes for splicing factors.

    PubMed

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

    2010-04-01

    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.

  2. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    PubMed

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program.

  3. Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.

    PubMed Central

    Aroian, R V; Levy, A D; Koga, M; Ohshima, Y; Kramer, J M; Sternberg, P W

    1993-01-01

    The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs. Images PMID:8417357

  4. MAASE: An alternative splicing database designed for supporting splicing microarray applications

    PubMed Central

    ZHENG, CHRISTINA L.; KWON, YOUNG-SOO; LI, HAI-RI; ZHANG, KUI; COUTINHO-MANSFIELD, GABRIELA; YANG, CANZHU; NAIR, T. MURLIDHARAN; GRIBSKOV, MICHAEL; FU, XIANG-DONG

    2005-01-01

    Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication. PMID:16251387

  5. SAW: A Method to Identify Splicing Events from RNA-Seq Data Based on Splicing Fingerprints

    PubMed Central

    Ning, Kang; Fermin, Damian

    2010-01-01

    Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons. PMID:20706591

  6. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  7. 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

  8. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

    PubMed Central

    Ng, Ashley Shu Mei; Ingham, Philip W.

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. PMID:27832146

  9. Synthetic chromosomes.

    PubMed

    Schindler, Daniel; Waldminghaus, Torsten

    2015-11-01

    What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes.

  10. SYNTHETIC LUBRICANTS

    DTIC Science & Technology

    of synthetic lubricants for use at low and high temperatures. The diesters of straight-chain dibasic acids lead the field of esters mutable as...lubricants for use at both low and high temperatures, because of their desirable combinations of properties and potentially good availability. Adipic ...azelaic, and sebacic acids are the most readily available dibasic acids suitable for ester lubricant production, while the petroleum derived Oxo alcohols

  11. Chemical syntheses of inhibitory substrates of the RNA-RNA ligation reaction catalyzed by the hairpin ribozyme.

    PubMed

    Massey, Archna P; Sigurdsson, Snorri Th

    2004-01-01

    The chemical syntheses of RNA oligomers containing modifications on the 5'-carbon of the 5'-terminal nucleoside for crystallographic and mechanistic studies of the hairpin ribozyme are reported. Phosphoramidites 4 and 8 were prepared and used in solid phase syntheses of RNA oligomers containing the sequence 5'-N'UCCUCUCC, where N' indicates either 5'-chloro-5'-deoxyguanosine or 5'-amino-5'-deoxyguanosine, respectively. A ribozyme ligation assay with the 5'-chloro- and 5'-amino-modified RNA oligomers demonstrated their inhibition of the hairpin-catalyzed RNA-RNA ligation reaction.

  12. Alternative splicing in cancer: implications for biology and therapy.

    PubMed

    Chen, J; Weiss, W A

    2015-01-02

    Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

  13. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed

    Miao, Zhichao; Adamiak, Ryszard W; Antczak, Maciej; Batey, Robert T; Becka, Alexander J; Biesiada, Marcin; Boniecki, Michał J; Bujnicki, Janusz; Chen, Shi-Jie; Cheng, Clarence Yu; Chou, Fang-Chieh; Ferré-D'Amaré, Adrian R; Das, Rhiju; Dawson, Wayne K; Feng, Ding; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanisław; Geniesse, Caleb; Kappel, Kalli; Kladwang, Wipapat; Krokhotin, Andrey; Łach, Grzegorz E; Major, François; Mann, Thomas H; Magnus, Marcin; Pachulska-Wieczorek, Katarzyna; Patel, Dinshaw J; Piccirilli, Joseph A; Popenda, Mariusz; Purzycka, Katarzyna J; Ren, Aiming; Rice, Greggory M; Santalucia, John; Sarzynska, Joanna; Szachniuk, Marta; Tandon, Arpit; Trausch, Jeremiah J; Tian, Siqi; Wang, Jian; Weeks, Kevin M; Williams, Benfeard; Xiao, Yi; Xu, Xiaojun; Zhang, Dong; Zok, Tomasz; Westhof, Eric

    2017-01-30

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

  19. Communication between RNA folding domains revealed by folding of circularly permuted ribozymes.

    PubMed

    Lease, Richard A; Adilakshmi, Tadepalli; Heilman-Miller, Susan; Woodson, Sarah A

    2007-10-12

    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 degrees 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 degrees 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 T(1) 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.

  20. 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

  1. 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

  2. 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.

  3. Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme.

    PubMed

    Bouchard, Patricia; Lacroix-Labonté, Julie; Desjardins, Geneviève; Lampron, Philipe; Lisi, Véronique; Lemieux, Sébastien; Major, François; Legault, Pascale

    2008-04-01

    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 (31)P and (15)N 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.

  4. 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

  5. Lessons from non-canonical splicing

    PubMed Central

    Ule, Jernej

    2016-01-01

    Recent improvements in experimental and computational techniques used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons, and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimises their potential to disrupt gene expression. While non-canonical splicing can lead to aberrant transcripts that cause many diseases, we also explain how it can be exploited for new therapeutic strategies. PMID:27240813

  6. RNA-splicing endonuclease structure and function.

    PubMed

    Calvin, K; Li, H

    2008-04-01

    The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes.

  7. HOLLYWOOD: a comparative relational database of alternative splicing.

    PubMed

    Holste, Dirk; Huo, George; Tung, Vivian; Burge, Christopher B

    2006-01-01

    RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

  8. Synthetic wisdom.

    PubMed

    Kitcher, Philip

    2016-11-01

    Wisdom is a special kind of virtue. It is not to be identified with any outstanding cognitive ability-like having a prodigious memory or knowing a lot. Rather it consists in seeing what is most important and most valuable, either within a particular domain or in life as a whole. In the life of a wise person, that insight should be accompanied by traits of character, enabling the person to pursue what is seen as valuable. Viewing wisdom as a capacity for synthetic understanding, I argue for the need for philosophy, even at a time when all of us have much to learn from the sciences.

  9. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2001-10-01

    2001. Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of Caenorhabditis elegans . 11... Caenorhabditis 36 Lorson, C.L. et al. (1999) A single nucleotide in the Biol. 77, 277-291 elegans . Nature 402, 835-838 SMN gene regulates splicing and is...terminate (Birse et al., 1998; Proudfoot, 2000), mutation of the poly(A) signal resulted in the accumulation to high levels in the nuclear fraction of

  10. 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

    Bonneau, Eric; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

    2015-09-01

    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 Mg(2+)-binding sites within this structure using Mn(2+)-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 Mg(2+) 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 Mg(2+) 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.

  11. 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

  12. Targeting RNA-splicing for SMA treatment.

    PubMed

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

    The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease.

  13. Synthetic chloroplasts

    SciTech Connect

    Calvin, M.

    1980-06-01

    The principal function of the chloroplast is to capture solar quanta and to store them in some stable form. We are in the process of trying to construct a totally synthetic system that would simulate some of the reactions of the two photosystems which occur in natural chloroplasts. Toward this end, we have demonstrated a number of the reactions required in separated systems. We have shown that it is possible to transfer electrons across an insulating membrane barrier with a surfactant photosensitizer. Others have shown, and we have confirmed, that it is possible to collect the two electrons necessary for the generation of molecular hydrogen on a heterogeneous catalyst suspended in water and similarly to collect the four holes on another heterogeneous catalyst suspended in water for the generation of molecular oxygen. A synthesis of some of these molecular catalysts for both these purposes is underway, with some partial success. When these partial reactions are assembled in a system, the resulting synthetic chloroplasts will not resemble the natural entity in detailed construction as they will contain no protein.

  14. 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

  15. 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

  16. 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

    2015-12-07

    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.

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

    PubMed

    Dagenais, Pierre; Girard, Nicolas; Bonneau, Eric; Legault, Pascale

    2017-04-06

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

  18. 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

  19. 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

  20. Synthesis of 2'-C-alpha-difluoromethylarauridine and its 3'-O-phosphoramidite incorporation into a hammerhead ribozyme.

    PubMed

    Dunkel, M; Reither, V

    1999-03-08

    The 2'-C-difluoromethylated nucleoside 4 was synthesized starting from uridine. 4 was then converted to the 3'-O-phosphoramidite derivative 5 and was incorporated into a hammerhead ribozyme (7). The cleavage characteristics of the modified oligonucleotide have been analysed.

  1. 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.

  2. 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

  3. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis.

    PubMed

    Gaffarogullari, Ece Cazibe; Krause, André; Balbo, Jessica; Herten, Dirk-Peter; Jäschke, Andres

    2013-12-01

    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.

  11. Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes

    PubMed Central

    Amontov, Sergey; Jäschke, Andres

    2006-01-01

    Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these principles. The only natural allosteric ribozyme reported to date catalyzes an RNA cleavage reaction, and so do almost all artificial systems. RNA has, however, been shown to accelerate a much wider range of chemical reactions. Here we report that RNA catalysts for organic reactions can be put under the stringent control of effector molecules by straight-forward rational design. This approach uses known RNA sequences with catalytic and ligand-binding properties, and exploits weakly conserved sequence elements and available structural information to induce the formation of alternative, catalytically inactive structures. The potential and general applicability is demonstrated by the design of three different systems in which the rate of a catalytic carbon–carbon bond forming reaction is positively regulated up to 2100-fold by theophylline, tobramycin and a specific mRNA sequence, respectively. Although smaller in size than a tRNA, all three ribozymes show typical features of allosteric metabolic enzymes, namely high rate acceleration and tight allosteric regulation. Not only do these findings demonstrate RNA's power as a catalyst, but also highlight on RNA's capabilities as signaling components in regulatory networks. PMID:16990253

  12. Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes.

    PubMed

    Amontov, Sergey; Jäschke, Andres

    2006-01-01

    Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these principles. The only natural allosteric ribozyme reported to date catalyzes an RNA cleavage reaction, and so do almost all artificial systems. RNA has, however, been shown to accelerate a much wider range of chemical reactions. Here we report that RNA catalysts for organic reactions can be put under the stringent control of effector molecules by straight-forward rational design. This approach uses known RNA sequences with catalytic and ligand-binding properties, and exploits weakly conserved sequence elements and available structural information to induce the formation of alternative, catalytically inactive structures. The potential and general applicability is demonstrated by the design of three different systems in which the rate of a catalytic carbon-carbon bond forming reaction is positively regulated up to 2100-fold by theophylline, tobramycin and a specific mRNA sequence, respectively. Although smaller in size than a tRNA, all three ribozymes show typical features of allosteric metabolic enzymes, namely high rate acceleration and tight allosteric regulation. Not only do these findings demonstrate RNA's power as a catalyst, but also highlight on RNA's capabilities as signaling components in regulatory networks.

  13. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM.

    PubMed

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-11-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

  14. 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

  15. Synthetic Botany.

    PubMed

    Boehm, Christian R; Pollak, Bernardo; Purswani, Nuri; Patron, Nicola; Haseloff, Jim

    2017-02-28

    Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.

  16. 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/).

  17. 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

    2015-01-09

    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.

  18. 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

  19. 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'.

  20. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    PubMed

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  1. 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.

  2. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  3. Alternative splicing modulates stem cell differentiation.

    PubMed

    Fu, Ru-Huei; Liu, Shih-Ping; Ou, Chen-Wei; Yu, Hsiu-Hui; Li, Kuo-Wei; Tsai, Chang-Hai; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2009-01-01

    Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell-cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.

  4. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  5. Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

    DTIC Science & Technology

    2009-04-01

    CT stably knocked down to undetectable levels using anti-CT hammerhead ribozymes [25]. Salmon CT (BAChem, Torrance, CA) was used at a physiologic...called CTR-, derived from PC-3M cells after anti- CT receptor ribozyme knockdown of CTR[18]. CTR- cells have very low levels of CD44v protein[4

  6. Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

    DTIC Science & Technology

    2009-10-01

    CT-cells have endog- enous CT stably knocked down to undetectable levels using anti-CT hammerhead ribozymes [25]. Salmon CT (BAChem, Torrance, CA) was...and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knock- down of CTR[18]. CTR-cells have very low levels of CD44v

  7. NMR structure of the 5' splice site in the group IIB intron Sc.ai5γ--conformational requirements for exon-intron recognition.

    PubMed

    Kruschel, Daniela; Skilandat, Miriam; Sigel, Roland K O

    2014-03-01

    A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.

  8. Synthetic Astrobiology

    NASA Technical Reports Server (NTRS)

    Rothschild, Lynn J.

    2017-01-01

    "Are we alone?" is one of the primary questions of astrobiology, and whose answer defines our significance in the universe. Unfortunately, this quest is hindered by the fact that we have only one confirmed example of life, that of earth. While this is enormously helpful in helping to define the minimum envelope for life, it strains credulity to imagine that life, if it arose multiple times, has not taken other routes. To help fill this gap, our lab has begun using synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - as an enabling technology. One theme, the "Hell Cell" project, focuses on creating artificial extremophiles in order to push the limits for Earth life, and to understand how difficult it is for life to evolve into extreme niches. In another project, we are re-evolving biotic functions using only the most thermodynamically stable amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids.

  9. Synthetic Astrobiology

    NASA Technical Reports Server (NTRS)

    Rothschild, Lynn J.

    2015-01-01

    'Are we alone?' is one of the primary questions of astrobiology, and whose answer defines our significance in the universe. Unfortunately, this quest is hindered by the fact that we have only one confirmed example of life, that of earth. While this is enormously helpful in helping to define the minimum envelope for life, it strains credulity to imagine that life, if it arose multiple times, has not taken other routes. To help fill this gap, our lab has begun using synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - as an enabling technology. One theme, the "Hell Cell" project, focuses on creating artificial extremophiles in order to push the limits for Earth life, and to understand how difficult it is for life to evolve into extreme niches. In another project, we are re-evolving biotic functions using only the most thermodynamically stable amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids.

  10. 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

  11. The 5'-AG5 CC-3' Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G-Quadruplex.

    PubMed

    Kulikov, Katharina; Nozinovic, Senada; Kath-Schorr, Stephanie

    2017-03-13

    An unusually thermostable G-quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G-quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.

  12. 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.

  13. 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

  14. The 20S Proteasome Splicing Activity Discovered by SpliceMet

    PubMed Central

    Textoris-Taube, Kathrin; Janek, Katharina; Keller, Christin; Henklein, Petra; Kloetzel, Peter Michael; Zaikin, Alexey

    2010-01-01

    The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected. PMID:20613855

  15. 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.

  16. Analysis of differential splicing suggests different modes of short-term splicing regulation

    PubMed Central

    Topa, Hande; Honkela, Antti

    2016-01-01

    Motivation: Alternative splicing is an important mechanism in which the regions of pre-mRNAs are differentially joined in order to form different transcript isoforms. Alternative splicing is involved in the regulation of normal physiological functions but also linked to the development of diseases such as cancer. We analyse differential expression and splicing using RNA-sequencing time series in three different settings: overall gene expression levels, absolute transcript expression levels and relative transcript expression levels. Results: Using estrogen receptor α signaling response as a model system, our Gaussian process-based test identifies genes with differential splicing and/or differentially expressed transcripts. We discover genes with consistent changes in alternative splicing independent of changes in absolute expression and genes where some transcripts change whereas others stay constant in absolute level. The results suggest classes of genes with different modes of alternative splicing regulation during the experiment. Availability and Implementation: R and Matlab codes implementing the method are available at https://github.com/PROBIC/diffsplicing. An interactive browser for viewing all model fits is available at http://users.ics.aalto.fi/hande/splicingGP/ Contact: hande.topa@helsinki.fi or antti.honkela@helsinki.fi Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307611

  17. Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

    PubMed

    Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.

  18. Targeting MDM4 Splicing in Cancers

    PubMed Central

    Bardot, Boris; Toledo, Franck

    2017-01-01

    MDM4, an essential negative regulator of the P53 tumor suppressor, is frequently overexpressed in cancer cells that harbor a wild-type P53. By a mechanism based on alternative splicing, the MDM4 gene generates two mutually exclusive isoforms: MDM4-FL, which encodes the full-length MDM4 protein, and a shorter splice variant called MDM4-S. Previous results suggested that the MDM4-S isoform could be an important driver of tumor development. In this short review, we discuss a recent set of data indicating that MDM4-S is more likely a passenger isoform during tumorigenesis and that targeting MDM4 splicing to prevent MDM4-FL protein expression appears as a promising strategy to reactivate p53 in cancer cells. The benefits and risks associated with this strategy are also discussed. PMID:28230750

  19. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  20. 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

  1. Systematic identification and analysis of exonic splicing silencers.

    PubMed

    Wang, Zefeng; Rolish, Michael E; Yeo, Gene; Tung, Vivian; Mawson, Matthew; Burge, Christopher B

    2004-12-17

    Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

  2. Efficient inhibition of beta-secretase gene expression in HEK293 cells by tRNAVal-driven and CTE-helicase associated hammerhead ribozymes.

    PubMed

    Nawrot, Barbara; Antoszczyk, Slawomir; Maszewska, Maria; Kuwabara, Tomoko; Warashina, Masaki; Taira, Kazunari; Stec, Wojciech J

    2003-10-01

    The beta-amyloid peptide (Abeta) is a major component of toxic amyloid plaques found in the brains of patients with Alzheimer's disease. Abeta is liberated by sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The level of Abeta depends directly on the hydrolytic activity of beta-secretase. Therefore, beta-secretase is an excellent target for drug design. An approach based on RNA-cleaving ribozymes was developed to control expression of beta-secretase. Two sites of mRNA coding beta-site APP cleaving enzyme were chosen as target sequences for endogenously delivered ribozymes. The ribozyme cassette was designed to constitute a catalytic hammerhead core and substrate recognition arms, flanked at the 5'-terminus by tRNAVal and at the 3'-terminus by constitutive transport element sequences. Ribozyme cassettes were cloned into a pUC19 plasmid and used for transient transfection of HEK293 cells. We demonstrate that such ribozymes efficiently inhibit beta-secretase gene expression at both the mRNA (up to 95%) and the protein (up to 90%) levels. Inhibition of beta-site APP cleaving enzyme activity directly influences the intra- and extracellular population of Abeta peptide. Therefore, such ribozymes may be considered as molecular tools for silencing the beta-secretase activity, and further, as therapeutic agents for anti-amyloid treatment.

  3. UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

    PubMed Central

    Kilani, A F; Liu, F

    1999-01-01

    RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes. PMID:10496224

  4. Processing and Translation Initiation of Non-long Terminal Repeat Retrotransposons by Hepatitis Delta Virus (HDV)-like Self-cleaving Ribozymes*

    PubMed Central

    Ruminski, Dana J.; Webb, Chiu-Ho T.; Riccitelli, Nathan J.; Lupták, Andrej

    2011-01-01

    Many non-long terminal repeat (non-LTR) retrotransposons lack internal promoters and are co-transcribed with their host genes. These transcripts need to be liberated before inserting into new loci. Using structure-based bioinformatics, we show that several classes of retrotransposons in phyla-spanning arthropods, nematodes, and chordates utilize self-cleaving ribozymes of the hepatitis delta virus (HDV) family for processing their 5′ termini. Ribozyme-terminated retrotransposons include rDNA-specific R2, R4, and R6, telomere-specific SART, and Baggins and RTE. The self-scission of the R2 ribozyme is strongly modulated by the insertion site sequence in the rDNA, with the most common insertion sequences promoting faster processing. The ribozymes also promote translation initiation of downstream open reading frames in vitro and in vivo. In some organisms HDV-like and hammerhead ribozymes appear to be dedicated to processing long and short interspersed elements, respectively. HDV-like ribozymes serve several distinct functions in non-LTR retrotransposition, including 5′ processing, translation initiation, and potentially trans-templating. PMID:21994949

  5. Experimental study of lap splice bolted connection

    NASA Astrophysics Data System (ADS)

    Zhao, Dehui; Tian, Lishan; Jiang, Wenqiang; An, Liqiang; Zhang, Ziyang

    2017-01-01

    The bolted connection is prone to slip under external load in the lattice transmission tower, which will affect the internal forces and deformation of tower. In order to better simulate the effect of bolt connection slippage on transmission tower, the load deformation relationship should be established. In this paper, the single lap splice bolt connection under tension load is tested and the load displacement curve is obtained. Furthermore, the existing model of single lap splice bolted connection is modified, which will plays an important role in the influence of the bolt slippage of the transmission lines towers more accurately and reasonably.

  6. 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....

  7. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  8. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Electrical connections or splices; suitability... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically...

  9. 30 CFR 75.514 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Electrical connections or splices; suitability... COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Electrical Equipment-General § 75.514 Electrical connections or splices; suitability. All electrical connections or splices...

  10. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Electrical connections or splices; suitability... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically...

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells.

    PubMed

    Jin, Seung-Gi; Mann, Jeffrey R

    2005-07-01

    The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.

  16. 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.

  17. Regulation of Alternative Splicing in Tumor Metastasis

    DTIC Science & Technology

    2000-10-01

    erythematosus and sarcoidosis . Arthritis Rheum. 41: 1505-15 10. Eldridge A.G., Y. Li, P.A. Sharp, and B.J. Blencowe. 1999. The SRml60/300 splicing coactivator...J. Hum. Genet. 59:279-286. erythematosus and sarcoidosis . Arthritis Rheum. 41:1505-1510. Matsumoto, K., K.M. Wassarman, and A.P. Wolffe. 1998. Nuclear

  18. 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.

  19. Emerging roles of BRCA1 alternative splicing.

    PubMed

    Orban, T I; Olah, E

    2003-08-01

    Germline mutations of the BRCA1 gene predispose individuals mainly to the development of breast and/or ovarian cancer. However, the exact function of the gene is still unclear, although the encoded proteins are involved in various cellular processes, including transcriptional regulation and DNA repair pathways. Several BRCA1 splice variants are found in different tissues, but in spite of intense investigations, their regulation and possible functions are poorly understood at the moment. This review summarises current knowledge on the roles of these splice variants and the mechanisms responsible for their formation. Because alternative splicing is now widely accepted as an important source of genetic diversity, elucidating the functions of the BRCA1 splice variants would help in the understanding of the exact role(s) of this tumour suppressor. This should help to resolve the current paradox that, despite its seemingly vital cellular functions, mutations of this gene are associated with tissue specific tumour formation predominantly in the breast and the ovary.

  20. Alternative splicing: a pivotal step between eukaryotic transcription and translation.

    PubMed

    Kornblihtt, Alberto R; Schor, Ignacio E; Alló, Mariano; Dujardin, Gwendal; Petrillo, Ezequiel; Muñoz, Manuel J

    2013-03-01

    Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

  1. Second order limit language in variants of splicing system

    NASA Astrophysics Data System (ADS)

    Ahmad, Muhammad Azrin; Sarmin, Nor Haniza; Heng, Fong Wan; Yusof, Yuhani

    2014-07-01

    The cutting and pasting processes that occur in DNA molecules have led to the formulation of splicing system. Since then, there are few models used to model the splicing system. The splicing language, which is the product of splicing system, can be categorized into two, namely the adult and limit language. In this research, limit language is extended to the second order limit language. Few problems are approached which lead to the formation of second order limit language which is then analyzed using various types of splicing system.

  2. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

    PubMed

    Listerman, Imke; Sapra, Aparna K; Neugebauer, Karla M

    2006-09-01

    Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

  3. Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.

    PubMed

    Liberman, Joseph A; Wedekind, Joseph E

    2011-06-01

    Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

  4. Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

    PubMed Central

    Carbonell, Alberto; Flores, Ricardo; Gago, Selma

    2011-01-01

    Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg2+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg2+. Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons. PMID:21097888

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Functional association between promoter structure and transcript alternative splicing.

    PubMed

    Cramer, P; Pesce, C G; Baralle, F E; Kornblihtt, A R

    1997-10-14

    It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

  11. 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

  12. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

    PubMed

    Klein, Daniel J; Wilkinson, Sara R; Been, Michael D; Ferré-D'Amaré, Adrian R

    2007-10-12

    The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

  13. 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

  14. The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme.

    PubMed

    Bingaman, Jamie L; Zhang, Sixue; Stevens, David R; Yennawar, Neela H; Hammes-Schiffer, Sharon; Bevilacqua, Philip C

    2017-04-01

    RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.

  15. 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

  16. Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus.

    PubMed

    Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M; Berenguer, José; Cedergren, Robert

    2002-04-01

    The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

  17. Multiple splicing defects in an intronic false exon.

    PubMed

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  18. 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

  19. Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA

    PubMed Central

    Schaal, Thomas D.; Maniatis, Tom

    1999-01-01

    We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3′ splice-site selection. PMID:9858550

  20. 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.

  1. 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

  2. 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

  3. Heritability of alternative splicing in the human genome

    PubMed Central

    Kwan, Tony; Benovoy, David; Dias, Christel; Gurd, Scott; Serre, David; Zuzan, Harry; Clark, Tyson A.; Schweitzer, Anthony; Staples, Michelle K.; Wang, Hui; Blume, John E.; Hudson, Thomas J.; Sladek, Rob; Majewski, Jacek

    2007-01-01

    Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5′ splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled. PMID:17671095

  4. 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.

  5. 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.

  6. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells.

    PubMed

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-09-01

    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.

  7. [EDAS, databases of alternatively spliced human genes].

    PubMed

    Nurtdinov, R N; Neverov, A D; Mal'ko, D B; Kosmodem'ianskiĭ, I A; Ermakova, E O; Ramenskiĭ, V E; Mironov, A A; Gel'fand, M S

    2006-01-01

    EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.

  8. 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.

  9. Synthetic biology, inspired by synthetic chemistry.

    PubMed

    Malinova, V; Nallani, M; Meier, W P; Sinner, E K

    2012-07-16

    The topic synthetic biology appears still as an 'empty basket to be filled'. However, there is already plenty of claims and visions, as well as convincing research strategies about the theme of synthetic biology. First of all, synthetic biology seems to be about the engineering of biology - about bottom-up and top-down approaches, compromising complexity versus stability of artificial architectures, relevant in biology. Synthetic biology accounts for heterogeneous approaches towards minimal and even artificial life, the engineering of biochemical pathways on the organismic level, the modelling of molecular processes and finally, the combination of synthetic with nature-derived materials and architectural concepts, such as a cellular membrane. Still, synthetic biology is a discipline, which embraces interdisciplinary attempts in order to have a profound, scientific base to enable the re-design of nature and to compose architectures and processes with man-made matter. We like to give an overview about the developments in the field of synthetic biology, regarding polymer-based analogs of cellular membranes and what questions can be answered by applying synthetic polymer science towards the smallest unit in life, namely a cell.

  10. 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

  11. 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

  12. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    PubMed

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells.

  13. 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.

  14. Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

    PubMed Central

    Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

    2007-01-01

    Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

  15. 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.

  16. Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition.

    PubMed

    Královicová, Jana; Vorechovsky, Igor

    2007-01-01

    Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.

  17. A multi-agent system simulating human splice site recognition.

    PubMed

    Vignal, L; Lisacek, F; Quinqueton, J; d'Aubenton-Carafa, Y; Thermes, C

    1999-06-15

    The present paper describes a method detecting splice sites automatically on the basis of sequence data and models of site/signal recognition supported by experimental evidences. The method is designed to simulate splicing and while doing so, track prediction failures, missing information and possibly test correcting hypotheses. Correlations between nucleotides in the splice site regions and the various elements of the acceptor region are evaluated and combined to assess compensating interactions between elements of the splicing machinery. A scanning model of the acceptor region and a model of interaction between the splicing complexes (exon definition model) are also incorporated in the detection process. Subsets of sites presenting deficiencies of several splice site elements could be identified. Further examination of these sites helps to determine lacking elements and refine models.

  18. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  19. Targeting Splicing in the Treatment of Human Disease

    PubMed Central

    Suñé-Pou, Marc; Prieto-Sánchez, Silvia; Boyero-Corral, Sofía; Moreno-Castro, Cristina; El Yousfi, Younes; Suñé-Negre, Josep Mª; Hernández-Munain, Cristina; Suñé, Carlos

    2017-01-01

    The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event. PMID:28245575

  20. Test and Analysis of Spliced DI-BSCCO HTS Tapes

    NASA Astrophysics Data System (ADS)

    Fetisov, S. S.; Sotnikov, D. V.; Radchenko, I. P.; Vysotsky, V. S.; Osabe, G.; Kinoshita, K.; Fujikami, J.; Kobayashi, S.; Yamazaki, K.

    For some applications, short unit lengths of HTS wires should be spliced if longer lengths are necessary and short unit lengths of HTS wires should be utilize by applying the splice technology to reduce the total wire cost in the application. The splice technology has been developed for DI-BSCCO Type HT-CA tapes by Sumitomo Electric and spliced tapes were tested in Russian Cable Institute. The test program included: measurements of splice's resistance, critical current anisotropy, thermo cycling tolerance, mechanical properties, overload tests and magnetization measurements. In the paper the results of tests are presented and discussed. The test results demonstrated that splices can be used for cable production if twisting and bending limitations are taken into account.

  1. HEXEvent: a database of Human EXon splicing Events.

    PubMed

    Busch, Anke; Hertel, Klemens J

    2013-01-01

    HEXEvent (http://hexevent.mmg.uci.edu) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

  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. Identification of common genetic variation that modulates alternative splicing.

    PubMed

    Hull, Jeremy; Campino, Susana; Rowlands, Kate; Chan, Man-Suen; Copley, Richard R; Taylor, Martin S; Rockett, Kirk; Elvidge, Gareth; Keating, Brendan; Knight, Julian; Kwiatkowski, Dominic

    2007-06-01

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  4. 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.

  5. 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

  6. 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

  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. 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

  9. 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

  10. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function

    NASA Astrophysics Data System (ADS)

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-03-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  11. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

    PubMed

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-04-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg(2+) concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  12. CHARMM force field parameters for simulation of reactive intermediates in native and thio-substituted ribozymes

    PubMed Central

    Mayaan, Evelyn; Moser, Adam; MacKerell, Alexander D.; York, Darrin M.

    2010-01-01

    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 Mg2+ 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 the CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges and 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 non-standard 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. PMID:17186477

  13. Mis-Spliced Lr34 Transcript Events in Winter Wheat

    PubMed Central

    Fang, Tilin; Carver, Brett F.; Hunger, Robert M.; Yan, Liuling

    2017-01-01

    Lr34 in wheat is a non-race-specific gene that confers resistance against multiple fungal pathogens. The resistant allele Lr34 and the susceptible allele Lr34s can be distinguished by three polymorphisms that cause alternation of deduced amino acid sequences of Lr34 at the protein level. In seedlings of a cultivar carrying the resistant Lr34r allele, only a portion (35%) of its transcripts was correctly spliced and the majority (65%) of its transcripts were incorrectly spliced due to multiple mis-splicing events. Lr34 mis-splicing events were also observed at adult plant age when this gene exerts its function. All of the mis-spliced Lr34r cDNA transcripts observed in this study resulted in a premature stop codon due to a shift of the open reading frame; hence, the mis-spliced Lr34r cDNAs were deduced to encode incomplete proteins. Even if a cultivar has a functional Lr34 gene, its transcripts might not completely splice in a correct pattern. These findings suggested that the partial resistance conferred by a quantitative gene might be due to mis-splicing events in its transcripts; hence, the resistance of the gene could be increased by eliminating or mutating regulators that cause mis-splicing events in wheat. PMID:28135317

  14. 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

  15. Evolutionary Insights into RNA trans-Splicing in Vertebrates.

    PubMed

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-03-10

    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.

  16. 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

  17. 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.

  18. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-05

    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.

  19. 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

  20. A divalent cation-dependent variant of the glmS ribozyme with stringent Ca(2+) selectivity co-opts a preexisting nonspecific metal ion-binding site.

    PubMed

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

    2017-03-01

    Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca(2+) in intracellular signaling, engineered ribozymes with stringent Ca(2+) selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmS(WT)) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glmS(AAA)) was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca(2+)-specific ribozyme (glmS(Ca)) evolved from glmS(AAA) that is >10,000 times more active in Ca(2+) than Mg(2+), is inactive in even 100 mM Mg(2+), and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca(2+) chelator such as EGTA. Because glmS(Ca) functions in physiologically relevant Ca(2+) concentrations, it can form the basis for intracellular sensors that couple Ca(2+) levels to RNA cleavage. Biochemical analysis of glmS(Ca) reveals that it has co-opted for selective Ca(2+) binding a nonspecific cation-binding site responsible for structural stabilization in glmS(WT) and glmS(AAA) Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.

  1. [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.

  2. [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.

  3. Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

    DTIC Science & Technology

    2008-04-01

    hammerhead ribozymes .25 Salmon CT (BAChem, Torrance, CA) was used at physiologic 50 nM dose14,16, which effectively alters CD44,6 or at 250 nM. To detect...receptor14), and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knockdown of CTR.18 CTR- cells have very low levels of

  4. 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

  5. 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

  6. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process.

    PubMed

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-07-24

    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.

  7. Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

    PubMed

    Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

    2014-01-01

    Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

    PubMed Central

    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 (–NH2) 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 MC, 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. PMID:26567314

  13. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  14. Cotranscriptional splicing efficiency differs dramatically between Drosophila and mouse.

    PubMed

    Khodor, Yevgenia L; Menet, Jerome S; Tolan, Michael; Rosbash, Michael

    2012-12-01

    Spliceosome assembly and/or splicing of a nascent transcript may be crucial for proper isoform expression and gene regulation in higher eukaryotes. We recently showed that cotranscriptional splicing occurs efficiently in Drosophila, but there are not comparable genome-wide nascent splicing data from mammals. To provide this comparison, we analyze a recently generated, high-throughput sequencing data set of mouse liver nascent RNA, originally studied for circadian transcriptional regulation. Cotranscriptional splicing is approximately twofold less efficient in mouse liver than in Drosophila, i.e., nascent intron levels relative to exon levels are ∼0.55 in mouse versus 0.25 in the fly. An additional difference between species is that only mouse cotranscriptional splicing is optimal when 5'-exon length is between 50 and 500 bp, and intron length does not correlate with splicing efficiency, consistent with exon definition. A similar analysis of intron and exon length dependence in the fly is more consistent with intron definition. Contrasted with these differences are many similarities between the two systems: Alternatively annotated introns are less efficiently spliced cotranscriptionally than constitutive introns, and introns of single-intron genes are less efficiently spliced than introns from multi-intron genes. The most striking common feature is intron position: Cotranscriptional splicing is much more efficient when introns are far from the 3' ends of their genes. Additionally, absolute gene length correlates positively with cotranscriptional splicing efficiency independently of intron location and position, in flies as well as in mice. The gene length and distance effects indicate that more "nascent time" gives rise to greater cotranscriptional splicing efficiency in both systems.

  15. 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-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy. 

  16. Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life

    PubMed Central

    Wong, J. Tze-Fei; Ng, Siu-Kin; Mat, Wai-Kin; Hu, Taobo; Xue, Hong

    2016-01-01

    The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets. PMID:26999216

  17. Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life.

    PubMed

    Wong, J Tze-Fei; Ng, Siu-Kin; Mat, Wai-Kin; Hu, Taobo; Xue, Hong

    2016-03-16

    The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets.

  18. Splice connector with internal heat transfer jacket

    DOEpatents

    Silva, Frank A.; Mayer, Robert W.

    1977-01-01

    A heat transfer jacket is placed over the terminal portions of the conductors of a pair of high voltage cables which are connected in a splice connection wherein a housing surrounds the connected conductor portions, the heat transfer jacket extending longitudinally between the confronting ends of a pair of adaptor sleeves placed upon the insulation of the cables to engage and locate the adaptor sleeves relative to one another, and laterally between the conductors and the housing to provide a path of relatively high thermal conductivity between the connected conductor portions and the housing.

  19. Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3′ end can be exported efficiently and specifically to the cytoplasm in mammalian cells

    PubMed Central

    Kuwabara, Tomoko; Warashina, Masaki; Koseki, Shiori; Sano, Masayuki; Ohkawa, Jun; Nakayama, Kazuhisa; Taira, Kazunari

    2001-01-01

    Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNAVal-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3′ end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5′ and 3′ ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells. PMID:11433023

  20. MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development

    PubMed Central

    Boutz, Paul L.; Chawla, Geetanjali; Stoilov, Peter; Black, Douglas L.

    2007-01-01

    Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3′ untranslated region (UTR), and a luciferase reporter carrying this 3′ UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program. PMID:17210790