Self-cleaving Ribozymes Research Articles

Scaling Catalytic Contributions of Small Self-Cleaving Ribozymes.

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

Rolling Circles as a Means of Encoding Genes in the RNA World.

The rolling circle mechanism found in viroids and some RNA viruses is a likely way that replication could have begun in the RNA World. Here, we consider simulations of populations of protocells, each containing multiple copies of rolling circle RNAs that can replicate non-enzymatically. The mechanism requires the presence of short self-cleaving ribozymes such as hammerheads, which can cleave and re-circularize RNA strands. A rolling circle must encode a hammerhead and the complement of a hammerhead, so that both plus and minus strands can cleave. Thus, the minimal functional length is twice the length of the hammerhead sequence. Selection for speed of replication will tend to reduce circles to this minimum length. However, if sequence errors occur when copying the hammerhead sequence, this prevents cleavage at one point, but still allows cleavage on the next passage around the rolling circle. Thus, there is a natural doubling mechanism that creates strands that are multiple times the length of the minimal sequence. This can provide space for the origin of new genes with beneficial functions. We show that if a beneficial gene appears in this new space, the longer sequence with the beneficial function can be selected, even though it replicates more slowly. This provides a route for the evolution of longer circles encoding multiple genes.

Predicting higher-order mutational effects in an RNA enzyme by machine learning of high-throughput experimental data.

Ribozymes are RNA molecules that catalyze biochemical reactions. Self-cleaving ribozymes are a common naturally occurring class of ribozymes that catalyze site-specific cleavage of their own phosphodiester backbone. In addition to their natural functions, self-cleaving ribozymes have been used to engineer control of gene expression because they can be designed to alter RNA processing and stability. However, the rational design of ribozyme activity remains challenging, and many ribozyme-based systems are engineered or improved by random mutagenesis and selection (in vitro evolution). Improving a ribozyme-based system often requires several mutations to achieve the desired function, but extensive pairwise and higher-order epistasis prevent a simple prediction of the effect of multiple mutations that is needed for rational design. Recently, high-throughput sequencing-based approaches have produced data sets on the effects of numerous mutations in different ribozymes (RNA fitness landscapes). Here we used such high-throughput experimental data from variants of the CPEB3 self-cleaving ribozyme to train a predictive model through machine learning approaches. We trained models using either a random forest or long short-term memory (LSTM) recurrent neural network approach. We found that models trained on a comprehensive set of pairwise mutant data could predict active sequences at higher mutational distances, but the correlation between predicted and experimentally observed self-cleavage activity decreased with increasing mutational distance. Adding sequences with increasingly higher numbers of mutations to the training data improved the correlation at increasing mutational distances. Systematically reducing the size of the training data set suggests that a wide distribution of ribozyme activity may be the key to accurate predictions. Because the model predictions are based only on sequence and activity data, the results demonstrate that this machine learning approach allows readily obtainable experimental data to be used for RNA design efforts even for RNA molecules with unknown structures. The accurate prediction of RNA functions will enable a more comprehensive understanding of RNA fitness landscapes for studying evolution and for guiding RNA-based engineering efforts.

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis.

Electrostatic interactions are fundamental to RNA structure and function, and intimately influenced by solvation and the ion atmosphere. RNA enzymes, or ribozymes, are catalytic RNAs that are able to enhance reaction rates over a million-fold, despite having only a limited repertoire of building blocks and available set of chemical functional groups. Ribozyme active sites usually occur at junctions where negatively charged helices come together, and in many cases leverage this strained electrostatic environment to recruit metal ions in solution that can assist in catalysis. Similar strategies have been implicated in related artificially engineered DNA enzymes. Herein, we apply Poisson-Boltzmann, 3D-RISM, and molecular simulations to study a set of metal-dependent small self-cleaving ribozymes (hammerhead, pistol, and Varkud satellite) as well as an artificially engineered DNAzyme (8-17) to examine electrostatic features and their relation to the recruitment of monovalent and divalent metal ions important for activity. We examine several fundamental roles for these ions that include: (1) structural integrity of the catalytically active state, (2) pKa tuning of residues involved in acid-base catalysis, and (3) direct electrostatic stabilization of the transition state via Lewis acid catalysis. Taken together, these examples demonstrate how RNA electrostatics orchestrates the site-specific and territorial binding of metal ions to play important roles in catalysis.

QRNAstruct: a method for extracting secondary structural features of RNA via regression with biological activity.

Recent technological advances have enabled the generation of large amounts of data consisting of RNA sequences and their functional activity. Here, we propose a method for extracting secondary structure features that affect the functional activity of RNA from sequence–activity data. Given pairs of RNA sequences and their corresponding bioactivity values, our method calculates position-specific structural features of the input RNA sequences, considering every possible secondary structure of each RNA. A Ridge regression model is trained using the structural features as feature vectors and the bioactivity values as response variables. Optimized model parameters indicate how secondary structure features affect bioactivity. We used our method to extract intramolecular structural features of bacterial translation initiation sites and self-cleaving ribozymes, and the intermolecular features between rRNAs and Shine–Dalgarno sequences and between U1 RNAs and splicing sites. We not only identified known structural features but also revealed more detailed insights into structure–activity relationships than previously reported. Importantly, the datasets we analyzed here were obtained from different experimental systems and differed in size, sequence length and similarity, and number of RNA molecules involved, demonstrating that our method is applicable to various types of data consisting of RNA sequences and bioactivity values.

A life of research on circular RNAs and ribozymes: towards the origin of viroids, deltaviruses and life

The first examples of circular RNAs (circRNAs) were reported in the ‘70s as a family of minimal infectious agents of flowering plants; the viroids and viral satellites of circRNA. In some cases, these small circular genomes encode self-cleaving RNA motifs or ribozymes, including an exceptional circRNA infecting not plants but humans: the Hepatitis Delta Virus. Autocatalytic ribozymes not only allowed to propose a common rolling-circle replication mechanism for all these subviral agents, but also a tentative link with the origin of life as molecular fossils of the so-called RNA world. Despite the weak biologic connection between angiosperm plants and the human liver, diverse scientists, and most notably Ricardo Flores, firmly supported an evolutionary relationship between plant viroids and human deltavirus agents. The tireless and inspiring work done by Ricardo's lab in the field of infectious circRNAs fuelled multiple hypotheses for the origin of these entities, allowing advances in other fields, from eukaryotic circRNAs to small ribozymes in genomes from all life kingdoms. The recent discovery of a plethora of viral-like circRNAs with ribozymes in disparate biological samples may finally allow us to connect plant and animal subviral agents, confirming again that Ricardo's eye for science was always a keen eye.

Structural Insights Into Tautomeric Dynamics in Nucleic Acids and in Antiviral Nucleoside Analogs.

DNA (2′-deoxyribonucleic acid) and RNA (ribonucleic acid) play diverse functional roles in biology and disease. Despite being comprised primarily of only four cognate nucleobases, nucleic acids can adopt complex three-dimensional structures, and RNA in particular, can catalyze biochemical reactions to regulate a wide variety of biological processes. Such chemical versatility is due in part to the phenomenon of nucleobase tautomerism, whereby the bases can adopt multiple, yet distinct isomeric forms, known as tautomers. For nucleobases, tautomers refer to structural isomers that differ from one another by the position of protons. By altering the position of protons on nucleobases, many of which play critical roles for hydrogen bonding and base pairing interactions, tautomerism has profound effects on the biochemical processes involving nucleic acids. For example, the transient formation of minor tautomers during replication could generate spontaneous mutations. These mutations could arise from the stabilization of mismatches, in the active site of polymerases, in conformations involving minor tautomers that are indistinguishable from canonical base pairs. In this review, we discuss the evidence for tautomerism in DNA, and its consequences to the fidelity of DNA replication. Also reviewed are RNA systems, such as the riboswitches and self-cleaving ribozymes, in which tautomerism plays a functional role in ligand recognition and catalysis, respectively. We also discuss tautomeric nucleoside analogs that are efficacious as antiviral drug candidates such as molnupiravir for coronaviruses and KP1212 for HIV. The antiviral efficacy of these analogs is due, in part, to their ability to exist in multiple tautomeric forms and induce mutations in the replicating viral genomes. From a technical standpoint, minor tautomers of nucleobases are challenging to identify directly because they are rare and interconvert on a fast, millisecond to nanosecond, time scale. Nevertheless, many approaches including biochemical, structural, computational and spectroscopic methods have been developed to study tautomeric dynamics in RNA and DNA systems, and in antiviral nucleoside analogs. An overview of these methods and their applications is included here.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq.

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5' fragment with a 2',3' cyclic phosphate (2',3' cP) and a 3' fragment with a 5' hydroxyl (5' OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5' OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3' phosphate of the adapter and then ligates it directly to RNAs with 5' OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3' twister ribozyme fragment from E.coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5' OH termini from total RNA.

Further in-silico Evidence for the Evolution of Transfer RNA from Ribozymes

Since the transfer RNA (tRNA) of Archaea is believed to be the oldest nucleic acid and domain of microorganisms, respectively, on Earth, its dependency on protein endonuclease enzymes for processing is thought to be the result of coevolution of tRNA intron motifs with tRNA endonuclease architecture. Another school of thought, however, suggests that ribozymes, ancient RNA-only catalysts which perform many of the same functions as present-day protein enzymes, were responsible for the processing of archaeal tRNA, much in the same manner that they currently still do in bacterial tRNA. This hypothesis has recently gained even more support with the in-silico discovery of vestigial aminoacylating ribozyme nucleotide sequences in some of the oldest modern-day Archaea. To that end, an in-silico study was performed in an attempt to find evidence of vestigial magnesium-dependent hammerhead self-cleaving ribozyme sequence motifs in archaeal tRNA.

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes.

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

CyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2ʹ,3ʹ cyclic phosphates and 5ʹ hydroxyl ends

ABSTRACT Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5′-fragment with a 2′,3′-cyclic phosphate and a 3′-fragment with a 5′-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5′-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3′-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms.

Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.

Herein, Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation levels in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy.

Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches.

A small molecule-responsive self-cleaving ribozyme (aptazyme) embedded in the untranslated region of an mRNA functions as a riboswitch that allows chemical regulation of gene expression in mammalian cells. Aptazymes are engineered by fusing a self-cleaving ribozyme with an RNA aptamer that recognizes a small molecule so that the ribozyme is either activated or inhibited in the presence of the small molecule. However, the variety of aptamers, ribozymes, and aptazyme design strategies suitable for mammalian riboswitch applications is still limited. This work focuses on a new ribozyme scaffold for engineering aptazymes and riboswitches that function in mammalian cells. We investigated circularly permuted variants of the pistol ribozyme class (CPP) as a synthetic ribozyme scaffold for mammalian riboswitch applications. Through semirational design and high-throughput screening, we designed guanine and tetracycline activated riboswitches based on three distinct aptazyme architectures, resulting in riboswitches with ON/OFF ratios as high as 8.6. Our work adds CPP to the limited ribozyme scaffold toolbox for mammalian synthetic biology applications and highlights the opportunities in exploring ribozymes beyond natural motifs.

Identification of over 200-fold more hairpin ribozymes than previously known in diverse circular RNAs.

Self-cleaving ribozymes are catalytic RNAs that cut themselves at a specific inter-nucleotide linkage. They serve as a model of RNA catalysis, and as an important tool in biotechnology. For most of the nine known structural classes of self-cleaving ribozymes, at least hundreds of examples are known, and some are present in multiple domains of life. By contrast, only four unique examples of the hairpin ribozyme class are known, despite its discovery in 1986. We bioinformatically predicted 941 unique hairpin ribozymes of a different permuted form from the four previously known hairpin ribozymes, and experimentally confirmed several diverse predictions. These results profoundly expand the number of natural hairpin ribozymes, enabling biochemical analysis based on natural sequences, and suggest that a distinct permuted form is more biologically relevant. Moreover, all novel hairpins were discovered in metatranscriptomes. They apparently reside in RNA molecules that vary both in size—from 381 to 5170 nucleotides—and in protein content. The RNA molecules likely replicate as circular single-stranded RNAs, and potentially provide a dramatic increase in diversity of such RNAs. Moreover, these organisms have eluded previous attempts to isolate RNA viruses from metatranscriptomes—suggesting a significant untapped universe of viruses or other organisms hidden within metatranscriptome sequences.

Trans ligation of RNAs to generate hybrid circular RNAs using highly efficient autocatalytic transcripts.

Circular RNAs are useful entities for various biotechnology applications, such as templating translation and binding or sequestering miRNA and RNA binding proteins. Circular RNA as highly resistant to degradation in cells and are more long-lived than linear RNAs. Here, we describe a method for intracellular trans ligation of RNA transcripts that can generate hybrid circular RNAs. These hybrid circular RNAs comprise two separate RNA that are covalently linked by ligation to form a circular RNA. By incorporating self-cleaving ribozymes at each site of ligation, trans ligation of the transcripts occurs in mammalian cells with no additional material. We provide a protocol for designing and testing trans ligation of transcripts and demonstrate detection of hybrid circular RNAs using fluorescence microscopy.

Hunting for human ribozymes.

An approach based on enrichment of 5′ hydroxylated RNAs reveals a new self-cleaving ribozyme that maps to a very long non-protein-coding RNA in simians. The ribozyme is active only among hominin sequences, suggesting a recent acquisition of the activity.

Hovlinc is a recently evolved class of ribozyme found in human lncRNA.

Although naturally occurring catalytic RNA molecules-ribozymes-have attracted a great deal of research interest, very few have been identified in humans. Here, we developed a genome-wide approach to discovering self-cleaving ribozymes and identified a naturally occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to an unidentified class of small, self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path, from its appearance between ~130 and ~65 million years ago (Ma), to acquiring self-cleavage activity very recently, ~13-10 Ma, in the common ancestors of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within a long noncoding RNA belonging to a class of very long intergenic noncoding RNAs. The presence of a catalytic RNA enzyme in lncRNA creates the possibility that these transcripts could function by carrying catalytic RNA domains.

Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa

ABSTRACT Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme – an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.

Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation.

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.

Comprehensive Sequence-to-Function Mapping of Ligand-Dependent RNA Catalysis

Massively-parallel, quantitative measurements of biochemical activity have the potential to revolutionize our understanding of biomolecular sequence-structure-function relationships. We report the development of a novel RNA array assay which enables such measurements to be performed on self-cleaving ribozymes, allowing determination of real-time cleavage kinetics of tens of thousands of sequence variants in a single experiment. We applied this methodology to the glmS ribozyme riboswitch, which performs self-cleavage in a ligand-dependent manner in order to regulate bacterial cell wall synthesis. We determined the cleavage rates of all single and double mutants of this RNA under a range of regulatory ligand concentrations, enabling the calculation of kcat and KM for active variants. This trove of quantitative kinetic parameters across the mutational landscape enabled a number of further analyses. Comparing single-mutant activities with naturally-occurring sequence variation at each position, we found that conservation is driven by cleavage rate, and not by ligand binding affinity. Comprehensive double-mutant measurements permitted a global analysis of rescue interactions, which revealed the functional relevance of several known tertiary contacts, as well as providing evidence for a number of compensatory secondary and tertiary structural rearrangements. Double-mutant kinetics and rescue interactions also provided a combined structural and functional mapping of the ribozyme which successfully identified the full secondary structure at nucleotide resolution, while additionally revealing relative structural and catalytic contributions of individual residues. We believe that our approach will be broadly applicable and extensible, enabling a deeper understanding of sequence-structure-function relationships across a variety of functional RNAs.