Ribozyme Research Articles

First unravelling of the hidden and intricate evolutionary history of a bacterial group II intron family.

Bacterial group II introns are large RNA enzymes that self-splice from primary transcripts. Following excision, they can invade various DNA target sites using RNA-based mobility pathways. As fast evolving retromobile elements, that move between genetic loci within and across species, their evolutionary history was proved difficult to study and infer. Here we identified several homologs of Ll.LtrB, the model group II intron from Lactococcus lactis, and traced back their evolutionary relationship through phylogenetic analyses. Our data demonstrate that the Ll.LtrB homologs in Lactococci originate from a single and recent lateral transfer event of Ef.PcfG from Enterococcus faecalis. We also show that these introns disseminated in Lactococci following recurrent episodes of independent mobility events in conjunction with occurences of lateral transfer. Our phylogenies identified additional lateral transfer events from the environmental clade of the more diverged Lactococci introns to a series of low GC gram-positive bacterial species including E. faecalis. We also determined that functional intron adaptation occurred early in Lactococci following Ef.PcfG acquisition from E. faecalis and that two of the more diverged Ll.LtrB homologs remain proficient mobile elements despite the significant number of mutations acquired. This study describes the first comprehensive evolutionary history of a bacterial group II intron.

CRISPR/Cas9 system in hematopoietic stem cells: Basic research and clinical applications

Clustered regularly interspaced short palindromic repeats (CRISPR) approach adapted from the prokaryotic adaptive immune system against to pathogen attack is so valuable and promising tool for treatment of human malignant and non-malignant hematological disease and disorders through genome editing in hematopoietic stem cells (HSCs). Moreover, CRISPR/Cas9 approach is not only useful for therapeutic purposes; it is considerably preferred for the generation of in vitro and in vivo animal disease models. CRISPR/Cas9 approach has been developed for highly efficient on-target cleavage, and low off-target effect via delivery systems and manipulation of CRISPR components including single guide RNA (sgRNA) and Cas enzymes. In this review, we focused on the CRISPR/Cas9 system applications on hematopoietic stem cells in basic research and clinical area with basic research and clinical perspectives.

Ribozyme-mediated RNA synthesis and replication in a model Hadean microenvironment

Enzyme-catalyzed replication of nucleic acid sequences is a prerequisite for the survival and evolution of biological entities. Before the advent of protein synthesis, genetic information was most likely stored in and replicated by RNA. However, experimental systems for sustained RNA-dependent RNA-replication are difficult to realise, in part due to the high thermodynamic stability of duplex products and the low chemical stability of catalytic RNAs. Using a derivative of a group I intron as a model for an RNA replicase, we show that heated air-water interfaces that are exposed to a plausible CO2-rich atmosphere enable sense and antisense RNA replication as well as template-dependent synthesis and catalysis of a functional ribozyme in a one-pot reaction. Both reactions are driven by autonomous oscillations in salt concentrations and pH, resulting from precipitation of acidified dew droplets, which transiently destabilise RNA duplexes. Our results suggest that an abundant Hadean microenvironment may have promoted both replication and synthesis of functional RNAs.

Snapshots of the second-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM

Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self-splicing reactions, we determine the structures of L-16 Tetrahymena ribozyme in complex with a 5′-splice site analog product and a 3′-splice site analog substrate using cryo-EM. We solve six conformations from a single specimen, corresponding to different splicing intermediates after the first ester-transfer reaction. The structures reveal dynamics during self-splicing, including large conformational changes of the internal guide sequence and the J5/4 junction as well as subtle rearrangements of active-site metals and the hydrogen bond formed between the 2′-OH group of A261 and the N2 group of guanosine substrate. These results help complete a detailed structural and mechanistic view of this paradigmatic group I intron undergoing the second step of self-splicing.

Genomic Landscape and Potential Regulation of RNA Editing in Drug Resistance.

Adenosine-to-inosine RNA editing critically affects the response of cancer therapies. However, comprehensive identification of drug resistance-related RNA editing events and systematic understanding of how RNA editing mediates anticancer drug resistance remain unclear. Here, 7157 differential editing sites (DESs) are identified from 98127 informative RNA editing sites in tumor tissues, many of which are validated in cancer cell lines. Diverse editing patterns of DESs are discovered in resistant samples, which could not be fully explained by adenosine deaminase acting on RNA enzymes. Some RNA-binding proteins are identified that potentially regulate these editing events. Notably, the DESs are significantly enriched in 3'-untranslated regions (3'-UTRs). The impact of DESs in 3'-UTR on the microRNA (miRNA) regulations is explored, and some triplets (DES, miRNA, and gene) that may contribute to drug resistance are identified. In addition, it is determined that the functions of genes enriched with DESs are associated with drug resistance, such as apoptosis, drug metabolism, and DNA synthesis involved in DNA repair. An online resource (http://www.jianglab.cn/REDR/) to support convenient retrieval of DESs is also built. The findings reveal the landscape and potential regulatory mechanism of RNA editing in drug resistance, providing new therapeutic targets for reversing drug resistance.

Raumzeitliche Propagation eines minimalen katalytischen RNA‐Netzwerkes in GUV‐Protozellen durch Temperaturzyklen und Phasenseparation

AbstractKompartimentierung ist der Schlüssel zu vielen zellulären Prozessen und kritische Bedingung für minimales Leben. In Zellen sorgt eine komplexe Chemie dafür, Biomoleküle zur richtigen Zeit am richtigen Ort zusammenzubringen oder sie zu separieren. Durch die Selbstorganisation von Lipiden, Nukleinsäuren und Proteinen entstehen hochspezialisierte Mikroumgebungen mit funktionalen Grenzflächen und Schnittstellen. Wir stellen hier auf Basis von reversibler RNA‐basierter Flüssig‐Flüssig‐Phasenseparation (LLPS) in Riesenvesikeln (GUVs) ein effizientes System zur funktionalen Kompartimentierung vor. Dieses ist in der Lage, eine RNA‐basierte enzymatische Reaktion mithilfe von Frost‐Tau‐(FT)‐Zyklen innerhalb einer Population von GUVs zu propagieren. Wir zeigen, dass eine solche enzymatische Reaktion dadurch beschleunigt werden kann, dass die Reaktanden in der kondensierten RNA‐reichen Phase aufkonzentriert werden. Die gleichmäßige Verteilung von RNA‐Substraten im dekondensierten Zustand ermöglicht den Austausch zwischen den Vesikeln während des Einfrierens und Auftauens. In der vorliegenden Studie wurde untersucht, inwieweit ein minimales reversibles Phasenseparationssystem in Lipidvesikeln raumzeitliche Kontrolle in zyklischen Prozessen erlaubt, so wie es in Minimalzellen erforderlich wäre.

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation.

Compartmentalization is key to many cellular processes and a critical bottleneck of any minimal life approach. In cells, a complex chemistry is responsible for bringing together or separating biomolecules at the right place at the right time. Lipids, nucleic acids and proteins self-organize, thereby creating boundaries, interfaces and specialized microenvironments. Exploiting reversible RNA-based liquid-liquid phase separation (LLPS) inside giant unilamellar vesicles (GUVs), we present an efficient system capable of propagating an RNA-based enzymatic reaction across a population of GUVs upon freezing-thawing (FT) temperature cycles. We report that compartmentalization in the condensed RNA-rich phase can accelerate such an enzymatic reaction. In the decondensed state, RNA substrates become homogeneously dispersed, enabling content exchange between vesicles during freeze-thawing. This work explores how a minimal reversible phase separation system in lipid vesicles could help to implement spatiotemporal control in cyclic processes, as required for minimal cells.

A RNase P Ribozyme Inhibits Gene Expression and Replication of Hepatitis B Virus in Cultured Cells.

Hepatitis B virus (HBV), an international public health concern, is a leading viral cause of liver disease, such as hepatocellular carcinoma. Sequence-specific ribozymes derived from ribonuclease P (RNase P) catalytic RNA are being explored for gene targeting applications. In this study, we engineered an active RNase P ribozyme, M1-S-A, targeting the overlapping region of HBV S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA), all deemed essential for viral infection. Ribozyme M1-S-A cleaved the S mRNA sequence efficiently in vitro. We studied the effect of RNase P ribozyme on HBV gene expression and replication using the human hepatocyte HepG2.2.15 culture model that harbors an HBV genome and supports HBV replication. In these cultured cells, the expression of M1-S-A resulted in a reduction of more than 80% in both HBV RNA and protein levels and an inhibition of about 300-fold in the capsid-associated HBV DNA levels when compared to the cells that did not express any ribozymes. In control experiments, cells expressing an inactive control ribozyme displayed little impact on HBV RNA and protein levels, and on capsid-associated viral DNA levels. Our study signifies that RNase P ribozyme can suppress HBV gene expression and replication, implying the promise of RNase P ribozymes for anti-HBV therapy.

Periodic temperature changes drive the proliferation of self-replicating RNAs in vesicle populations

Growth and division of biological cells are based on the complex orchestration of spatiotemporally controlled reactions driven by highly evolved proteins. In contrast, it remains unknown how their primordial predecessors could achieve a stable inheritance of cytosolic components before the advent of translation. An attractive scenario assumes that periodic changes of environmental conditions acted as pacemakers for the proliferation of early protocells. Using catalytic RNA (ribozymes) as models for primitive biocatalytic molecules, we demonstrate that the repeated freezing and thawing of aqueous solutions enables the assembly of active ribozymes from inactive precursors encapsulated in separate lipid vesicle populations. Furthermore, we show that encapsulated ribozyme replicators can overcome freezing-induced content loss and successive dilution by freeze-thaw driven propagation in feedstock vesicles. Thus, cyclic freezing and melting of aqueous solvents – a plausible physicochemical driver likely present on early Earth – provides a simple scenario that uncouples compartment growth and division from RNA self-replication, while maintaining the propagation of these replicators inside new vesicle populations.

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Chemoproteomic discovery of a human RNA ligase

RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5′-PO4 and 3′-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5′-PO4 and 3′-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. These data provide the groundwork for establishing a human RNA repair pathway.

Melatonin Alleviates Chromium Toxicity in Maize by Modulation of Cell Wall Polysaccharides Biosynthesis, Glutathione Metabolism, and Antioxidant Capacity.

Melatonin, a pleiotropic regulatory molecule, is involved in the defense against heavy metal stress. Here, we used a combined transcriptomic and physiological approach to investigate the underlying mechanism of melatonin in mitigating chromium (Cr) toxicity in Zea mays L. Maize plants were treated with either melatonin (10, 25, 50 and 100 μM) or water and exposed to 100 μM K2Cr2O7 for seven days. We showed that melatonin treatment significantly decreased the Cr content in leaves. However, the Cr content in the roots was not affected by melatonin. Analyses of RNA sequencing, enzyme activities, and metabolite contents showed that melatonin affected cell wall polysaccharide biosynthesis, glutathione (GSH) metabolism, and redox homeostasis. During Cr stress, melatonin treatment increased cell wall polysaccharide contents, thereby retaining more Cr in the cell wall. Meanwhile, melatonin improved the GSH and phytochelatin contents to chelate Cr, and the chelated complexes were then transported to the vacuoles for sequestration. Furthermore, melatonin mitigated Cr-induced oxidative stress by enhancing the capacity of enzymatic and non-enzymatic antioxidants. Moreover, melatonin biosynthesis-defective mutants exhibited decreased Cr stress resistance, which was related to lower pectin, hemicellulose 1, and hemicellulose 2 than wild-type plants. These results suggest that melatonin alleviates Cr toxicity in maize by promoting Cr sequestration, re-establishing redox homeostasis, and inhibiting Cr transport from the root to the shoot.

Altered Mechanisms for Acid-Catalyzed RNA Cleavage and Isomerization Reactions Models.

RNA strand cleavage by 2'-O-transphosphorylation is catalyzed not only by numerous nucleolytic RNA enzymes (ribozymes) but also by hydroxide or hydronium ions. In experiments, both cleavage of the 5'-linked nucleoside and isomerization between 3',5'- and 2',5'-phosphodiesters occur under acidic conditions, while only the cleavage reaction is observed under basic conditions. An ab initio path-integral approach for simulating kinetic isotope effects is used to reveal the reaction mechanisms for RNA cleavage and isomerization reactions under acidic conditions. Moreover, the proposed mechanisms can also be combined through the experimental pH-rate profiles.

How did life become cellular?

How did life become cellular?

Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I.

RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5'-end (RNA I-5) resulted in an approximately twofold increase in the steady-state levels of RNA I-5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These results indicate that RNA I-5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5'-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5'-monophosphorylated end.

New insights into the centrosome-associated spliceosome components as regulators of ciliogenesis and tissue identity.

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids. Centrosomes are biomolecular condensates that play a crucial role in nuclear division, cytoskeletal remodeling, and cilia formation in animal cells. Spatial omics technology is providing new insights into the dynamic exchange of spliceosome components between the nucleus and the centrosome/cilium. Intriguingly, centrosomes are emerging as cytoplasmic sites for information storage, enriched with RNA molecules and RNA-processing proteins. Furthermore, growing evidence supports the view that nuclear spliceosome components assembled at the centrosome function as potential coordinators of splicing subprograms, pluripotency, and cell differentiation. In this article, we first discuss the current understanding of the centrosome/cilium complex, which controls both stem cell differentiation and pluripotency. We next explore the molecular mechanisms that govern splicing factor assembly and disassembly around the centrosome and examine how RNA processing pathways contribute to ciliogenesis. Finally, we discuss numerous unresolved compelling questions regarding the centrosome-associated spliceosome components and transcript variants within the cytoplasm as sources of RNA-based secondary messages in the regulation of cell identity and cell fate determination. This article is categorized under: RNA-Based Catalysis > RNA Catalysis in Splicing and Translation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Processing.

Dissociative Transition State in Hepatitis Delta Virus Ribozyme Catalysis.

Ribonucleases and small nucleolytic ribozymes are both able to catalyze RNA strand cleavage through 2'-O-transphosphorylation, provoking the question of whether protein and RNA enzymes facilitate mechanisms that pass through the same or distinct transition states. Here, we report the primary and secondary 18O kinetic isotope effects for hepatitis delta virus ribozyme catalysis that reveal a dissociative, metaphosphate-like transition state in stark contrast to the late, associative transition states observed for reactions catalyzed by specific base, Zn2+ ions, or ribonuclease A. This new information provides evidence for a discrete ribozyme active site design that modulates the RNA cleavage pathway to pass through an altered transition state.

The Interplay between RNA Editing Regulator ADAR1 and Immune Environment in Colorectal Cancer.

An abnormality in the regulation of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyzed adenosine-to-inosine (A-to-I) RNA editing, was closely associated with the highly aggressive biologic behavior and poor prognosis in many malignancies. In the present study, we aimed to investigate the relationship among transcript factors-microRNAs regulatory network, immune environment, and ADAR gene in colorectal carcinoma (CRC). The association among the expression levels of ADAR mRNA and copy number variation, methylation, and mutation status were comprehensively analyzed using cBioPortal, Wanderer, and UALCAN databases in CRC datasets. ADAR-transcript factors (TFs) and ADAR-miRNA regulation networks were constructed by Cistrome Cancer and miRWalk2.0, respectively. The full network and subnetworks for ADAR coexpression genes were constructed using the STRING database and visualized by the MCODE module of the Cytoscape app. The relationship between ADAR mRNA expression and the abundance of infiltrating immune cells in CRC patients was explored by the Tumor Immune Estimation Resource, CIBERSORT, and single-gene gene set enrichment analysis (GSEA). ADAR mRNA was elevated and was a cancer essential gene in CRC. ADAR mRNA and transcripts P110 were significantly elevated in CRC compared to normal controls. Low-level methylation in the promoter region and high copy number amplification of ADAR were responsible for high levels of ADAR mRNA expression. ADAR coexpression genes were mainly involved in immunoregulation, especially T-lymphocyte activation. Hub genes, including CD2, CD274, and FASLG, were also significantly upregulated in the ADAR-high group compared to the control group. Besides, M1 macrophages were enriched in the ADAR-high group compared to the control group. This study demonstrated that ADAR, a new essential gene, was involved in the immune regulator and was a novel immune treatment target in CRC.

Peptide conjugates with polyaromatic hydrocarbons can benefit the activity of catalytic RNAs.

Early stages of life likely employed catalytic RNAs (ribozymes) in many functions that are today filled by proteins. However, the earliest life forms must have emerged from heterogenous chemical mixtures, which included amino acids, short peptides, and many other compounds. Here we explored whether the presence of short peptides can help the emergence of catalytic RNAs. To do this, we conducted an in vitro selection for catalytic RNAs from randomized sequence in the presence of ten different peptides with a prebiotically plausible length of eight amino acids. This in vitro selection generated dozens of ribozymes, one of them with ∼900-fold higher activity in the presence of one specific peptide. Unexpectedly, the beneficial peptide had retained its N-terminal Fmoc protection group, and this group was required to benefit ribozyme activity. The same, or higher benefit resulted from peptide conjugates with prebiotically plausible polyaromatic hydrocarbons (PAHs) such as fluorene and naphthalene. This shows that PAH-peptide conjugates can act as potent cofactors to enhance ribozyme activity. The results are discussed in the context of the origin of life.

Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses.

Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.