Self-cleaving Ribozymes Research Articles

Reversible Gene Regulation in Mammalian Cells Using Riboswitch-Engineered Vesicular Stomatitis Virus Vector.

Synthetic riboswitches based on small molecule-responsive self-cleaving ribozymes (aptazymes) embedded in the untranslated regions (UTRs) allow chemical control of gene expression in mammalian cells. In this work, we used a guanine-responsive aptazyme to control transgene expression from a replication-incompetent vesicular stomatitis virus (VSV) vector. VSV is a nonsegmented, negative-sense, cytoplasmic RNA virus that replicates without DNA intermediates, and its applications for vaccines and oncolytic viral therapy are being explored. By inserting the guanine-activated ribozyme in the 3' UTRs of viral genes and transgenes, GFP expression from the VSV vector in mammalian cells was repressed by as much as 26.8-fold in the presence of guanine. Furthermore, we demonstrated reversible regulation of a transgene (secreted NanoLuc) by adding and withdrawing guanine from the medium over the course of 12 days. In summary, our riboswitch-controlled VSV vector allows robust, long-term, and reversible regulation of gene expression in mammalian cells without the risk of undesirable genomic integration.

The effect of adenine protonation on RNA phosphodiester backbone bond cleavage elucidated by deaza-nucleobase modifications and mass spectrometry.

The catalytic strategies of small self-cleaving ribozymes often involve interactions between nucleobases and the ribonucleic acid (RNA) backbone. Here we show that multiply protonated, gaseous RNA has an intrinsic preference for the formation of ionic hydrogen bonds between adenine protonated at N3 and the phosphodiester backbone moiety on its 5′-side that facilitates preferential phosphodiester backbone bond cleavage upon vibrational excitation by low-energy collisionally activated dissociation. Removal of the basic N3 site by deaza-modification of adenine was found to abrogate preferential phosphodiester backbone bond cleavage. No such effects were observed for N1 or N7 of adenine. Importantly, we found that the pH of the solution used for generation of the multiply protonated, gaseous RNA ions by electrospray ionization affects phosphodiester backbone bond cleavage next to adenine, which implies that the protonation patterns in solution are at least in part preserved during and after transfer into the gas phase. Our study suggests that interactions between protonated adenine and phosphodiester moieties of RNA may play a more important mechanistic role in biological processes than considered until now.

CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.

Hatchet ribozyme structure and implications for cleavage mechanism

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

Influence of a 3′ Terminal Ribozyme on AgoshRNA Biogenesis and Activity

Short hairpin RNAs (shRNAs) can induce gene silencing via the RNA interference (RNAi) mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2) protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshRNAs. Active anti-HIV AgoshRNAs were developed, but their RNAi activity was generally reduced compared with the matching shRNAs. In an attempt to further optimize the AgoshRNA design, we inserted several self-cleaving ribozymes at the 3′ terminus of the transcribed AgoshRNA and evaluated the impact on AgoshRNA processing and activity. The hepatitis delta virus (HDV) ribozyme is efficiently removed from the transcribed AgoshRNAs and generates a uniform 3′ overhang, which translates into the enhanced antiviral activity of these molecules.

One-Pot Production of RNA Nanoparticles via Automated Processing and Self-Assembly.

From the original sequencing of the human genome, it was found that about 98.5% of the genome did not code for proteins. Subsequent studies have now revealed that a much larger portion of the genome is related to short or long noncoding RNAs that regulate cellular activities. In addition to the milestones of chemical and protein drugs, it has been proposed that RNA drugs or drugs targeting RNA will become the third milestone in drug development ( Shu , Y. ; Adv. Drug Deliv. Rev. 2014 , 66 , 74 . ). Currently, the yield and cost for RNA nanoparticle or RNA drug production requires improvement in order to advance the RNA field in both research and clinical translation by reducing the multiple tedious manufacturing steps. For example, with 98.5% incorporation efficiency of chemical synthesis of a 100 nucleotide RNA strand, RNA oligos will result with 78% contamination of aborted byproducts. Thus, RNA nanotechnology is one of the remedies, because large RNA can be assembled from small RNA fragments via bottom-up self-assembly. Here we report the one-pot production of RNA nanoparticles via automated processing and self-assembly. The continuous production of RNA by rolling circle transcription (RCT) using a circular dsDNA template is coupled with self-cleaving ribozymes encoded in the concatemeric RNA transcripts. Production was monitored in real-time. Automatic production of RNA fragments enabled their assembly either in situ or via one-pot co-transcription to obtain RNA nanoparticles of desired motifs and functionalities from bottom-up assembly of multiple RNA fragments. In combination with the RNA nanoparticle construction process, a purification method using a large-scale electrophoresis column was also developed.

Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA

ABSTRACTThe recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3ʹ-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1.1 to 5.2 fold. We also demonstrate that this design can enhance CRISPR-based gene activation. We thus generated an improved CRISPR-Cpf1 system for more efficient gene editing and gene regulation.

Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

The number of self-cleaving small ribozymes has increased sharply in recent years. Advances have been made in describing these ribozymes in terms of four catalytic strategies: α describes in-line attack, β describes neutralization of the nonbridging oxygens, γ describes activation of the nucleophile, and δ describes stabilization of the leaving group. Current literature presents the rapid self-cleavage of the twister ribozyme in terms of all four of these classic catalytic strategies. Herein, we describe the nonspecific contribution of small molecules to ribozyme catalysis. At biological pH, the rate of the wild-type twister ribozyme is enhanced up to 5-fold in the presence of moderate buffer concentrations, similar to the 3-5-fold effects reported previously for buffer catalysis for protein enzymes. We observe this catalytic enhancement not only with standard laboratory buffers, but also with diverse biological small molecules, including imidazole, amino acids, and amino sugars. Brønsted plots suggest that small molecules assist in proton transfer, most likely with δ catalysis. Cellular small molecules provide a simple way to overcome the limited functional diversity of RNA and have the potential to participate in the catalytic mechanisms of many ribozymes in vivo.

SHAPE probing pictures Mg2+-dependent folding of small self-cleaving ribozymes.

Self-cleaving ribozymes are biologically relevant RNA molecules which catalyze site-specific cleavage of the phosphodiester backbone. Gathering knowledge of their three-dimensional structures is critical toward an in-depth understanding of their function and chemical mechanism. Equally important is collecting information on the folding process and the inherent dynamics of a ribozyme fold. Over the past years, Selective-2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) turned out to be a significant tool to probe secondary and tertiary interactions of diverse RNA species at the single nucleotide level under varying environmental conditions. Small self-cleaving ribozymes, however, have not been investigated by this method so far. Here, we describe SHAPE probing of pre-catalytic folds of the recently discovered ribozyme classes twister, twister-sister (TS), pistol and hatchet. The study has implications on Mg2+-dependent folding and reveals potentially dynamic residues of these ribozymes that are otherwise difficult to identify. For twister, TS and pistol ribozymes the new findings are discussed in the light of their crystal structures, and in case of twister also with respect to a smFRET folding analysis. For the hatchet ribozyme where an atomic resolution structure is not yet available, the SHAPE data challenge the proposed secondary structure model and point at selected residues and putative long-distance interactions that appear crucial for structure formation and cleavage activity.

Kinetic Parameters of trans Scission by Extended HDV-like Ribozymes and the Prospect for the Discovery of Genomic trans-Cleaving RNAs.

Hepatitis delta virus (HDV)-like ribozymes are self-cleaving catalytic RNAs with a widespread distribution in nature and biological roles ranging from self-scission during rolling-circle replication in viroids to co-transcriptional processing of eukaryotic retrotransposons, among others. The ribozymes fold into a double pseudoknot with a common catalytic core motif and highly variable peripheral domains. Like other self-cleaving ribozymes, HDV-like ribozymes can be converted into trans-acting catalytic RNAs by bisecting the self-cleaving variants at non-essential loops. Here we explore the trans-cleaving activity of ribozymes derived from the largest examples of the ribozymes (drz-Agam-2 family), which contain an extended domain between the substrate strand and the rest of the RNA. When this peripheral domain is bisected at its distal end, the substrate strand is recognized through two helices, rather than just one 7 bp helix common among the HDV ribozymes, resulting in stronger binding and increased sequence specificity. Kinetic characterization of the extended trans-cleaving ribozyme revealed an efficient trans-cleaving system with a surprisingly high KM', supporting a model that includes a recently proposed activation barrier related to the assembly of the catalytically competent ribozyme. The ribozymes also exhibit a very long koff for the products (∼2 weeks), resulting in a trade-off between sequence specificity and turnover. Finally, structure-based searches for the catalytic cores of these ribozymes in the genome of the mosquito Anopheles gambiae, combined with sequence searches for their putative substrates, revealed two potential ribozyme-substrate pairs that may represent examples of natural trans-cleaving ribozymes.

Circular RNAs Biogenesis in Eukaryotes Through Self-Cleaving Hammerhead Ribozymes.

Circular DNAs are frequent genomic molecules, especially among the simplest life beings, whereas circular RNAs have been regarded as weird nucleic acids in biology. Now we know that eukaryotes are able to express circRNAs, mostly derived from backsplicing mechanisms, and playing different biological roles such as regulation of RNA splicing and transcription, among others. However, a second natural and highly efficient pathway for the expression in vivo of circRNAshas been recently reported, which allows the accumulation of abundant small (100-1000nt) non-coding RNA circles through the participation of small self-cleaving RNAs or ribozymes called hammerhead ribozymes. These genome-encoded circRNAs with ribozymes seem to be a new family of smalland nonautonomous retrotransposable elements of plants and animals (so-called retrozymes), which will offer functional clues to the biology and evolution of circular RNA molecules as well as new biotechnological tools in this emerging field.

Elucidation of Catalytic Strategies of Small Nucleolytic Ribozymes From Comparative Analysis of Active Sites.

A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2'OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (β), neutralization of the O5' leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2'OH nucleophile by a guanine: one to acidify the 2'OH by hydrogen bond donation to it (γ') and one to acidify the 2'OH by releasing it from non-productive interactions by competitive hydrogen bonding (γ''). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ' and γ'' catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.

Highly motif- and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression

ABSTRACTRecent bioinformatics studies have demonstrated a wide-spread occurrence of the hammerhead ribozyme (HHR) and similar small endonucleolytic RNA motifs in all domains of life. It is becoming increasingly evident that such ribozyme motifs participate in important genetic processes in diverse organisms. Although the HHR motif has been studied for more than three decades, only little is known about the consequences of ribozyme activity on gene expression. In the present study we analysed eight different naturally occurring HHR sequences in diverse genetic and organismal contexts. We investigated the influence of active ribozymes incorporated into mRNAs in mammalian, yeast and bacterial expression systems. The experiments show an unexpectedly high degree of organism-specific variability of ribozyme-mediated effects on gene expression. The presented findings demonstrate that ribozyme cleavage profoundly affect gene expression. However, the extent of this effect varies and depends strongly on the respective genetic context. The fast-cleaving type 3 HHRs [CChMVd(-) and sLTSV(-)] generally tended to cause the strongest effects on intracellular gene expression. The presented results are important in order to address potential functions of naturally occurring ribozymes in RNA processing and post-transcriptional regulation of gene expression. Additionally, our results are of interest for biotechnology and synthetic biology approaches that aim at the utilisation of self-cleaving ribozymes as widely applicable tools for controlling genetic processes.

First Report of Grapevine hammerhead viroid-like RNA Infecting Grapevine (Vitis vinifera) in France

Grapevine hammerhead viroid-like RNA (GHVd) is a poorly characterized circular, single-stranded RNA molecule discovered through the bioinformatic analysis of small interfering RNAs of Italian grapevines of the Pinot noir variety (Wu et al., 2012). It is 375 nucleotides long and shares many structural features of viroids or of some small circular satellite RNA molecules. In particular, it has a predicted secondary structure similar to those of members of the Pelamoviroid genus with a branched region stabilized by a kissing-loop pseudoknot, and an extensively base-paired arm that encodes in its plus and minus strands self-cleaving hammerhead ribozymes (Wu et al., 2012; Giguere et al., 2014). Outside of conserved nucleotides in these hammerhead ribozymes, the primary sequence of GHVd has no significant similarities to any entries in Genbank. Besides the original Italian Pinot noir grapes, GHVd has been detected in Pinot noir plants of the 115 clone grown in California (Wu et al., 2012). Given that infectivit...

Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes.

The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage.

Rescue of high-specificity Cas9 variants using sgRNAs with matched 5\u2019 nucleotides

We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5’ nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.

Allosteric Modulation of the Faecalibacterium prausnitzii Hepatitis Delta Virus-like Ribozyme by Glucosamine 6-Phosphate: The Substrate of the Adjacent Gene Product.

Self-cleaving ribozymes were discovered 30 years ago and have been found throughout nature, from bacteria to animals, but little is known about their biological functions and regulation, particularly how cofactors and metabolites alter their activity. A hepatitis delta virus-like self-cleaving ribozyme maps upstream of a phosphoglucosamine mutase (glmM) open reading frame in the genome of the human gut bacterium Faecalibacterium prausnitzii. The presence of a ribozyme in the untranslated region of glmM suggests a regulation mechanism of gene expression. In the bacterial hexosamine biosynthesis pathway, the enzyme glmM catalyzes the isomerization of glucosamine 6-phosphate into glucosamine 1-phosphate. In this study, we investigated the effect of these metabolites on the co-transcriptional self-cleavage rate of the ribozyme. Our results suggest that glucosamine 6-phosphate, but not glucosamine 1-phosphate, is an allosteric ligand that increases the self-cleavage rate of drz-Fpra-1, providing the first known example of allosteric modulation of a self-cleaving ribozyme by the substrate of the adjacent gene product. Given that the ribozyme is activated by the glmM substrate, but not the product, this allosteric modulation may represent a potential feed-forward mechanism of gene expression regulation in bacteria.

Structure-based insights into self-cleavage by a four-way junctional twister-sister ribozyme

Here we report on the crystal structure and cleavage assays of a four-way junctional twister-sister self-cleaving ribozyme. Notably, 11 conserved spatially separated loop nucleotides are brought into close proximity at the ribozyme core through long-range interactions mediated by hydrated Mg2+ cations. The C62–A63 step at the cleavage site adopts a splayed-apart orientation, with flexible C62 directed outwards, whereas A63 is directed inwards and anchored by stacking and hydrogen-bonding interactions. Structure-guided studies of key base, sugar, and phosphate mutations in the twister-sister ribozyme, suggest contributions to the cleavage chemistry from interactions between a guanine at the active site and the non-bridging oxygen of the scissile phosphate, a feature found previously also for the related twister ribozyme. Our four-way junctional pre-catalytic structure differs significantly in the alignment at the cleavage step (splayed-apart vs. base-stacked) and surrounding residues and hydrated Mg2+ ions relative to a reported three-way junctional pre-catalytic structure of the twister-sister ribozyme.

Identification of 15 candidate structured noncoding RNA motifs in fungi by comparative genomics

BackgroundWith the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies.ResultsWe have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements.ConclusionsSystematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions.

Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes.

Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages.