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Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the pentatricopeptide repeat (PPR) co-expressed intron splicing-1 (PCIS1) factor, which was identified in silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The p ABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-sequencing and quantitative RT-PCR analyses of wild-type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory CI defects and altered plant growth. Our data further emphasize the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria. [ABSTRACT FROM AUTHOR]

Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience. [ABSTRACT FROM AUTHOR]

Sidney Altman’s discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P–related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere. [ABSTRACT FROM AUTHOR]

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degroncontrolled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages. [ABSTRACT FROM AUTHOR]

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression. [ABSTRACT FROM AUTHOR]

Discovery of impacts of RNA modifications in liver disease has accelerated recently, with research on N6-methyladenosine and 5-methylcytosine proving particularly fruitful. These findings have yielded potential biomarkers and therapeutics to inform prognosis and course of treatment for hepatocellular carcinoma and metabolic-associated steatohepatitis, and are likely to continue to yield results for other liver diseases. Despite these advances, a full mechanistic understanding of RNA modifications has been slow to advance owing to experimental limitations and the outsized impact of low levels of RNA modification. Both mechanistic studies looking at specific RNA modification sites and broad multi-omic studies using patient samples should be continued, with each type of study potentially informing the other and leading to novel insights into mechanisms and alternative effects of RNA-modifying enzymes and RNA modifications. RNA modifications have emerged as important mechanisms of gene regulation. Developmental, metabolic, and cell cycle regulatory processes are all affected by epitranscriptomic modifications, which control gene expression in a dynamic manner. The hepatic tissue is highly metabolically active and has an impressive ability to regenerate after injury. Cell proliferation, differentiation, and metabolism, which are all essential to the liver response to injury and regeneration, are regulated via RNA modification. Two such modifications, N 6-methyladenosine (m6A)and 5-methylcytosine (m5C), have been identified as prognostic disease markers and potential therapeutic targets for liver diseases. Here, we describe progress in understanding the role of RNA modifications in liver biology and disease and discuss specific areas where unexpected results could lead to improved future understanding. RNA modifications have emerged as important mechanisms of gene regulation. Developmental, metabolic, and cell cycle regulatory processes are all affected by epitranscriptomic modifications, which control gene expression in a dynamic manner. The hepatic tissue is highly metabolically active and has an impressive ability to regenerate after injury. Cell proliferation, differentiation, and metabolism, which are all essential to the liver response to injury and regeneration, are regulated via RNA modification. Two such modifications, N 6-methyladenosine (m6A)and 5-methylcytosine (m5C), have been identified as prognostic disease markers and potential therapeutic targets for liver diseases. Here, we describe progress in understanding the role of RNA modifications in liver biology and disease and discuss specific areas where unexpected results could lead to improved future understanding. [ABSTRACT FROM AUTHOR]

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes – theta ribozymes – potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes – we provide 1753 unique examples – highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3'-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages. The diverse functional roles of ribozymes (RNAs with enzymatic activity) continue to be uncovered. Here, the authors identify and characterize a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes – termed Theta ribozymes -, which they show process viral tRNA transcripts, and appearing crucial for lytic gene expression in recoded phages. [ABSTRACT FROM AUTHOR]

Mitochondria are important organelles that provide energy for the life of cells. Group II introns are usually found in the mitochondrial genes of land plants. Correct splicing of group II introns is critical to mitochondrial gene expression, mitochondrial biological function, and plant growth and development. Ancestral group II introns are self-splicing ribozymes that can catalyze their own removal from pre-RNAs, while group II introns in land plant mitochondria went through degenerations in RNA structures, and thus they lost the ability to self-splice. Instead, splicing of these introns in the mitochondria of land plants is promoted by nuclear- and mitochondrial-encoded proteins. Many proteins involved in mitochondrial group II intron splicing have been characterized in land plants to date. Here, we present a summary of research progress on mitochondrial group II intron splicing in land plants, with a major focus on protein splicing factors and their probable functions on the splicing of mitochondrial group II introns. [ABSTRACT FROM AUTHOR]

Transcription represents a central aspect of gene expression with RNA polymerase machineries (RNA Pol) driving the synthesis of RNA from DNA template molecules. In eukaryotes, a total of three RNA Pol enzymes generate the plethora of RNA species and RNA Pol II is the one transcribing all protein-coding genes. A high number of cis- and trans-acting factors orchestrates RNA Pol II-mediated transcription by influencing the chromatin recruitment, activation, elongation, and/or termination steps. The levels of DNA accessibility, defining open-euchromatin versus close-heterochromatin, delimits RNA Pol II activity as well as the encounter with other factors acting on chromatin such as the DNA replication or DNA repair machineries. The stage of the cell cycle highly influences RNA Pol II activity with mitosis representing the major challenge. In fact, there is a massive inhibition of transcription during the mitotic entry coupled with chromatin dissociation of most of the components of the transcriptional machinery. Mitosis, as a consequence, highly compromises the transcriptional memory and the perpetuation of cellular identity. Once mitosis ends, transcription levels immediately recover to define the cell fate and to safeguard the proper progression of daughter cells through the cell cycle. In this review, we evaluate our current understanding of the transcriptional repression associated with mitosis with a special focus on the molecular mechanisms involved, on the potential function behind the general repression, and on the transmission of the transcriptional machinery into the daughter cells. We finally discuss the contribution that errors in the inheritance of the transcriptional machinery across mitosis might play in stem cell aging. [ABSTRACT FROM AUTHOR]

Small non‐coding RNAs called microRNAs (miRNAs) control the expression of genes post‐transcriptionally. Their correlation with commercial economic traits including milk, meat and egg production, as well as their effective role in animal productivity, fertility, embryo survival and disease resistance, make them significant in livestock research. The miRNAs exhibit distinct spatial and temporal expression patterns, offering insights into their functional roles within cells and tissues. Aberrant miRNA production can disrupt vital cellular processes and genetic networks, contributing to conditions like metabolic disorders and viral diseases. These short RNA molecules are present in extracellular fluids, displaying remarkable stability against RNA degradation enzymes and extreme environmental conditions. miRNAs preservation is facilitated through packaging in lipid vesicles or complex formation with RNA‐binding proteins. Numerous studies have illuminated the roles of miRNAs in diverse physiological processes, including embryonic stem cell differentiation, haematopoietic stem cell proliferation and differentiation and the coordinated development of organ systems. The integration of miRNA profiling, next‐generation sequencing and bioinformatics analysis paves the way for transformative advancements in livestock research and industry. The present review underscores the applications of miRNAs in livestock, showcasing their potential to improve breeding strategies, diagnose diseases and enhance our understanding of fundamental biological processes. [ABSTRACT FROM AUTHOR]

Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Overexpression of the predicted RNA-binding protein Ssr1238 in the cyanobacterium Synechocystis 6803 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript is derived from gene ssl3177, which encodes a central enzyme in cell wall remodelling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and could be involved in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the B. subtilis YlxR protein functions as an RNase P modulator (RnpM), extending its proposed role to the phylum cyanobacteria, and suggesting additional functionalities. [ABSTRACT FROM AUTHOR]

Self-cleaving ribozymes are versatile tools for synthetic biologists when it comes to controlling gene expression. Up to date, 12 different classes are known, and over the past decades more and more details about their structure, cleavage mechanisms and natural environments have been uncovered. However, when these motifs are applied to mammalian gene expression constructs, the outcome can often be unexpected. A variety of factors, such as surrounding sequences and positioning of the ribozyme influences the activity and hence performance of catalytic RNAs. While some information about the efficiency of individual ribozymes (each tested in specific contexts) is known, general trends obtained from standardized, comparable experiments are lacking, complicating decisions such as which ribozyme to choose and where to insert it into the target mRNA. In many cases, application-specific optimization is required, which can be very laborious. Here, we systematically compared different classes of ribozymes within the 3'-UTR of a given reporter gene. We then examined position-dependent effects of the best-performing ribozymes. Moreover, we tested additional variants of already widely used hammerhead ribozymes originating from various organisms. We were able to identify functional structures suited for aptazyme design and generated highly efficient hammerhead ribozyme variants originating from the human genome. The present dataset will aide decisions about how to apply ribozymes for affecting gene expression as well as for developing ribozyme-based switches for controlling gene expression in human cells. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbon (PAH) pollutants and microbiome products converge on the aryl hydrocarbon receptor (AhR) to redirect selective rapid adherence of isolated bone marrow (BM) cells. In young adult mice, Cyp1b1-deficiency and AhR activation by PAH, particularly when prolonged by Cyp1a1 deletion, produce matching gene stimulations in these BM cells. Vascular expression of Cyp1b1 lowers reactive oxygen species (ROS), suppressing NF-κB/RelA signaling. PAH and allelic selectivity support a non-canonical AhR participation, possibly through RelA. Genes stimulated by Cyp1b1 deficiency were further resolved according to the effects of Cyp1b1 and Cyp1a1 dual deletions (DKO). The adherent BM cells show a cluster of novel stimulations, including select developmental markers; multiple re-purposed olfactory receptors (OLFR); and α-Defensin, a microbial disruptor. Each one connects to an enhanced specific expression of the catalytic RNA Pol2 A subunit, among 12 different subunits. Mesenchymal progenitor BMS2 cells retain these features. Cyp1b1-deficiency removes lymphocytes from adherent assemblies as BM-derived mesenchymal stromal cells (BM-MSC) expand. Cyp1b1 effects were cell-type specific. In vivo, BM-MSC Cyp1b1 expression mediated PAH suppression of lymphocyte progenitors. In vitro, OP9-MSC sustained these progenitors, while Csf1 induced monocyte progenitor expansion to macrophages. Targeted Cyp1b1 deletion (Cdh5-Cre; Cyp1b1fl/fl) established endothelium control of ROS that directs AhR-mediated suppression of B cell progenitors. Monocyte Cyp1b1 deletion (Lyz2-Cre; Cyp1b1fl/fl) selectively attenuated M1 polarization of expanded macrophages, but did not enhance effects on basal M2 polarization. Thus, specific sources of Cyp1b1 link to AhR and to an OLFR network to provide BM inflammatory modulation via diverse microbiome products. [ABSTRACT FROM AUTHOR]

Kazal-type serine protease inhibitors (KaSPI) play important roles in insect growth, development, digestion, metabolism and immune defence. In this study, based on the transcriptome of Mythimna separata , the cDNA sequence of MsKaSPI with Kazal domain was uploaded to GenBank (MN931651). Spatial and temporal expression analysis showed that MsKaSPI was expressed at different developmental stages and different tissues, and it was induced by 20-hydroxyecdysone in third-instar larvae of M. separata. After 24 h infection by Beauveria bassiana , the expression level of MsKaSPI and the corresponding MsKaSPI content were significantly up-regulated, being 6.42-fold and 1.91-fold to the control group, respectively, while the activities of serine protease, trypsin and chymotrypsin were inhibited. After RNA interference interfered with MsKaSPI for 6 h, the expression decreased by 73.44%, the corresponding content of MsKaSPI protein decreased by 55.66% after 12 h, and the activities of serine protease and trypsin were significantly enhanced. Meanwhile, both the larval and pupal stages of M. separata were prolonged, the weights were reduced and the number of eggs per female decreased by 181. Beauveria bassiana infection also increased the mortality of MsKaSPI -silenced M. separata by 18.96%. These prove MsKaSPI can not only result in slow growth and low fecundity of M. separata by regulating the activity of related protease, but also participate in the resistance to pathogenic fungi by regulating the serine protease inhibitor content and the activities of related serine protease. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is an extremely malignant pulmonary vascular disease of unknown etiology. ADAR1 is an RNA editing enzyme that converts adenosine in RNA to inosine, thereby affecting RNA expression. However, the role of ADAR1 in PH development remains unclear. In the present study, we investigated the biological role and molecular mechanism of ADAR1 in PH pulmonary vascular remodeling. Overexpression of ADAR1 aggravated PH progression and promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs). Conversely, inhibition of ADAR1 produced opposite effects. High-throughput whole transcriptome sequencing showed that ADAR1 was an important regulator of circRNAs in PH. CircCDK17 level was significantly lowered in the serum of PH patients. The effects of ADAR1 on cell cycle progression and proliferation were mediated by circCDK17. ADAR1 affects the stability of circCDK17 by mediating A-to-I modification at the A5 and A293 sites of circCDK17 to prevent it from m1A modification. We demonstrate for the first time that ADAR1 contributes to the PH development, at least partially, through m1A modification of circCDK17 and the subsequent PASMCs proliferation. Our study provides a novel therapeutic strategy for treatment of PH and the evidence for circCDK17 as a potential novel marker for the diagnosis of this disease. The expression of ADAR1 is elevated under hypoxic condition. ADAR1 binds to circCDK17 and reduces its expression through inhibition of its m1A modification. This further reduces the ubiquitination levels of PCNA and promotes the proliferation of PASMCs, leading to pulmonary vascular remodeling. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair. [ABSTRACT FROM AUTHOR]

High-speed allosteric hammerhead ribozymes can be engineered to distinguish well between a perfectly matching effector and the nucleic acid sequences with a few mismatches under physiologically relevant conditions. Such ribozymes can be designed to control the expression of exogenous mRNAs and can be used to develop new gene therapies, including anticancer treatments. The in vivo selection of such ribozymes is a complicated and lengthy procedure with no guarantee of success. Thus, in silico selection of high-speed ribozymes can be employed using secondary RNA structure computation based on the partition function of the RNA folding in combination with random search algorithms. This approach has already been proven very accurate in designing allosteric hammerhead ribozymes. Herein, we present two programs for the computational design of allosteric ribozymes sensing randomized oligonucleotides based on the extended version of the hammerhead ribozyme. A Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with NOT logic function (GHOST-NOT) and a Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with YES logic function (GHOST-YES) for computational design of high-speed allosteric ribozymes are described. The allosteric hammerhead ribozymes had a high self-cleavage rate of about 1.8 per minute and were very selective in sensing an effector sequence. [ABSTRACT FROM AUTHOR]

Objective：To discuss the expression of circs_EFCAB2 in epileptic cell model in vitro, and to clarify its possible mechanism. Methods：The magnesium-free epilepsy cell model (model group) was constructed in the human neuroblastoma LA-N-5 cells, and the cells untreated with magnesium-free extracellular fluid were regarded as control group. Real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression level of circ_EFCAB2 mRNA in the cells in two groups. The cells were didived into ribonuclease (RNase) R digestion group and RNase R undigestion group. RNA enzyme digestion experiment and RT-qPCR method were used to detect the expression levels of circ_ EFCAB2 and linear EFCAB2 mRNA in the cells in two groups; nuclear cytoplasmic separation experiment was used to detect the expression levels of circle_EFCAB2 mRNA in the epileptic cells; CCK-8 method was used to detect the proliferation activities of the cells in two groups; flow cytometry was used to detect the apoptotic rate and percentages of the cells at different cell cycles in two groups. Results：The cells in model group showed spontaneous high-frequency epileptiform discharges, indicating the epilepsy cell model was successfully established. The RT-qPCR results showed that compared with control group, the expression level of circ_ EFCAB2 mRNA in the cells in model group was increased (P<0. 05). The RNA enzyme digestion experiment and RT-qPCR results showed that compared with RNase R undigestion group, the expression level of linear EFCAB2 mRNA in the cells in RNase R digestion group was decreased (P<0. 01). The nuclear cytoplasmic separation experiment results showed that there was no statistically significant difference in the expression level of circ_ EFCAB2 mRNA in the epileptic cells between the cytoplasm and nucleus of the epileptic cells (P>0. 05). The CCK-8 results showed that at 72 h after transfection, compared with control group, the proliferation activity of the cells in model group was significantly decreased (P<0. 01). The flow cytometry analysis results showed that compared with control group, the apoptotic rate of the cells in model group was significantly increased (P<0. 01); compared with control group, the percentage of the cells at S phase in model group was significantly decreased (P< 0. 01), and the percentage of the cells at G1+G2 phase was significantly increased (P< 0. 01). Conclusion：The upregulated circ_ EFCAB2 may play a role in the pathogenesis of epilepsy by inhibiting the cell proliferation, promoting the apoptosis, and blocking the cell cycles. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92–94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy. [ABSTRACT FROM AUTHOR]

Ageing is a conserved and unavoidable biological process characterized by progressive decline of physiological functions with time. Despite constituting the greatest risk factor for most human diseases, little is known about the molecular mechanisms driving the ageing process. More than 170 chemical RNA modifications, also known as the epitranscriptome, decorate eukaryotic coding and non-coding RNAs and have emerged as novel regulators of RNA metabolism, modulating RNA stability, translation, splicing or non-coding RNA processing. Studies on short-lived organisms such as yeast or worms connect mutations on RNA modifying enzymes with lifespan changes, and dysregulation of the epitranscriptome has been linked to age-related diseases and ageing hallmarks themselves in mammals. Moreover, transcriptome-wide analyses are starting to reveal changes in messenger RNA modifications in neurodegenerative diseases and in the expression of some RNA modifiers with age. These studies are starting to put the focus on the epitranscriptome as a potential novel regulator of ageing and lifespan, and open new avenues for the identification of targets to treat age-related diseases. In this review, we discuss the connection between RNA modifications and the enzymatic machinery regulating their deposition in coding and non-coding RNAs, and ageing and hypothesize about the potential role of RNA modifications in the regulation of other ncRNAs playing a key role in ageing, such as transposable elements and tRNA fragments. Finally, we reanalyze available datasets of mouse tissues during ageing and report a wide transcriptional dysregulation of proteins involved in the deposition, removal or decoding of several of the best-known RNA modifications. [ABSTRACT FROM AUTHOR]

Since the late 1970s, our understanding of the functions of RNA has been redefined by the discovery of introns and splicing, catalytic RNAs, RNA species not involved in translation, small regulatory RNAs, and pervasive transcription. High depth sequencing revealed the presence of large numbers of long noncoding (lnc)RNAs expressed intronically, intergenically, and antisense to protein-coding genes. The expression of lncRNAs is often cell type specific and many are multi-exonic and extensively alternatively spliced, indicating a modular structure. LncRNAs are involved in many aspects of cell biology, the control of differentiation and development, and disease. New research is discovering the association of lncRNAs with chromatin-modifying enzymes and phase-separated domains, and suggests that many, if not most, lncRNAs emanate from enhancers. RNA has long been regarded primarily as the intermediate between genes and proteins. It was a surprise then to discover that eukaryotic genes are mosaics of mRNA sequences interrupted by large tracts of transcribed but untranslated sequences, and that multicellular organisms also express many long 'intergenic' and antisense noncoding RNAs (lncRNAs). The identification of small RNAs that regulate mRNA translation and half-life did not disturb the prevailing view that animals and plant genomes are full of evolutionary debris and that their development is mainly supervised by transcription factors. Gathering evidence to the contrary involved addressing the low conservation, expression, and genetic visibility of lncRNAs, demonstrating their cell-specific roles in cell and developmental biology, and their association with chromatin-modifying complexes and phase-separated domains. The emerging picture is that most lncRNAs are the products of genetic loci termed 'enhancers', which marshal generic effector proteins to their sites of action to control cell fate decisions during development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Professor Cecília Maria Arraiano directs a research group named 'Control of Gene Expression' at Instituto de Tecnologia Química e Biológica, Universidade NOVA de Lisboa, Oeiras, Portugal. She started her scientific journey at the University of Lisbon, where she graduated in Biology, before completing her PhD in Genetics as a Fulbright‐Hays Fellow at the University of Georgia, Athens, USA. After a postdoc in the USA, she returned to Lisbon to establish her own lab. She has authored close to 200 publications mainly in the field of RNA degradation mechanisms, with a focus on enzymes and RNA chaperones that mediate RNA decay in microorganisms. She has received several prizes and is an active member of prestigious organizations. Namely, she is an EMBO member, Fellow of the European Academy of Microbiology, Fellow of the American Academy of Microbiology, and member of the Portuguese Academy of Sciences. In addition, Prof Arraiano has chaired the FEBS Working Group on Women in Science from 2014 to 2022. In this fascinating interview, she discusses her research, her experience working in the USA and Portugal, and the importance of initiatives to support women in science. [ABSTRACT FROM AUTHOR]

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2 -knockdown (Adar2 -KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2 -KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer. [Display omitted] • RNA editing enzyme ADAR2 is involved in chemoresistance in breast cancer cells. • Downregulation of ADAR2 increases P-glycoprotein, promoting doxorubicin efflux. • Downregulation of ADAR2 induces circHif1a expression. • CircHif1a inhibits miR-195a-3p, resulting in the increased P-glycoprotein expression. [ABSTRACT FROM AUTHOR]

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme-RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme-RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme-RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme-RNA interactions in connecting intermediary metabolism and gene expression. [ABSTRACT FROM AUTHOR]

Background: Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results: We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions: The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics. [ABSTRACT FROM AUTHOR]

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1β and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages. Alqassim et al find that RNA editing, known to be induced by IFN-1 in macrophages by the enzyme APOBEC3A, is required for the transcriptional, pro-inflammatory and metabolic responses that drive M1 macrophage polarization. APOBEC3A-mediated editing is also induced by IFN-1 exposure in tumor-associated macrophages isolated from ovarian cancer-related ascites fluid, pointing to a wide role for APOBEC3A in macrophages. [ABSTRACT FROM AUTHOR]

RNA capping and decapping tightly coordinate with transcription, translation, and RNA decay to regulate gene expression. Proteins in the DXO/Rai1 family have been implicated in mRNA decapping and decay, and mammalian DXO was recently found to also function as a decapping enzyme for NAD+‐capped RNAs (NAD‐RNA). The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein, AtDXO1. Here we show that AtDXO1 possesses both NAD‐RNA decapping activity and 5ʹ‐3ʹ exonuclease activity but does not hydrolyze the m7G cap. The atdxo1 mutation increased the stability of NAD‐RNAs and led to pleiotropic phenotypes, including severe growth retardation, pale color, and multiple developmental defects. Transcriptome profiling analysis showed that the atdxo1 mutation resulted in upregulation of defense‐related genes but downregulation of photosynthesis‐related genes. The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation. However, the various phenotypes associated with the atdxo1 mutant could be complemented by an enzymatically inactive AtDXO1. The atdxo1 mutation apparently enhances post‐transcriptional gene silencing by elevating levels of siRNAs. Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic molecular functions in mediating RNA processing and decay. [ABSTRACT FROM AUTHOR]

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5′ vs. 3′ untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N 6-methyladenosine (m6A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m6A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients. [Display omitted] • METTL3 promotes the expression of CXCL1, CXCL5, and CCL20 via c-Myc in NSCLC • METTL3 mediates PD-L1 mRNA m6A modification, resulting in destabilizing PD-L1 in NSCLC • METTL3 inhibition improves anti-PD-1 therapy in syngeneic murine LLC tumor model • METTL3 deficiency enhances PD-1 blockade in KP and orthotopic mouse lung tumor models Yu et al. report inhibiting METTL3 reprograms an inflamed tumor microenvironment that renders anti-PD-1 therapy more effective in several preclinical mouse NSCLC tumor models, highlighting the combination of METTL3 inhibitor with PD-1/PD-L1 immune-checkpoint blockade as a promising therapeutic strategy for NSCLC patients. [ABSTRACT FROM AUTHOR]

• Gene regulation can be achieved with very little coding space. • Direct interaction with the RNA leaves only minor off-target effects. • No additionally expressed proteins are necessary for gene regulation. • Small molecules as ligands show no toxicity to the cell. Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast. [ABSTRACT FROM AUTHOR]

Group II introns are ribozymes which can catalyze its own splicing and relegate itself. They share common structural features and are evolutionarily related to eukaryotic spliceosome. Hence, group II introns are an excellent model system for understanding the mechanism of RNA-splicing in gene expression. Recent advancement of structural studies has provided x-ray structures of group II intron at different stages (Pre-hydrolytic, post-hydrolytic and free intron) of splicing pathway and revealed heteronuclear metal ion cluster (two potassium; K1 and K2, two magnesium; M1 and M2) in the active site as common structural feature of group II introns. It is believed that these four metal ions are very crucial for catalysis. K + is very important for the function and buffers containing only Na + can destroy the function of group II introns. Despite the available 3D structures of different stages of self-splicing, the energetic origin of K + selectivity over Na + is not known. However, these structures provide sufficiently good models for directly computing the energetics of K + vs Na + discrimination that is required for understanding self-splicing. Here, we report extensive (∼550 ns) of structure-based molecular dynamics free energy simulations that quantitatively estimated K1 vs Na1 discrimination. We show that the strength of discrimination varies along the self-splicing pathway. The free and pre-hydrolytic states are highly selective for K1 and the selectivity is lost in the post-hydrolytic state. The simulations further reveal that Na1 in the free and pre-hydrolytic state is trapped with an unsatisfied first coordination shell in the active site, which is responsible for large discrimination. The Na1 in the active site of post-hydrolytic state allow water entry and satisfy the Na1 bonding requirement, resulting in low discrimination. The results not only give insights into the experimentally unresolved Na1 bound complexes but also provide the link between structures and computed energetics. [ABSTRACT FROM AUTHOR]

Abstract: Generation of conditional mutants in Trypanosoma brucei can be done by the use of RNA interference (RNAi). However, RNAi frequently produces off target effects. Here, we present an alternative strategy in which the glmS ribozyme is inserted in the C‐terminal region of one allele of a GOI and effectively knocks it down in response to the presence of glucosamine in the culture medium. Using several endogenous genes, we show that the glmS ribozyme cleaves the mRNA in vivo leading to reduction in mRNA and protein expression following glucosamine treatment in both T. brucei procyclic and bloodstream forms. Glucosamine‐induced ribozyme activation can be rapidly reversed by removing the inducer. In summary, the glmS ribozyme could be used as a tool to study essential genes in T. brucei. [ABSTRACT FROM AUTHOR]

Accuracy of transcription is essential for productive gene expression, and the past decade has brought new understanding of the mechanisms ensuring transcription fidelity. The discovery of a new catalytic domain, the Trigger Loop, revealed that RNA polymerase can actively choose the correct substrates. Also, the intrinsic proofreading activity was found to proceed via a ribozyme-like mechanism, whereby the erroneous nucleoside triphosphate (NTP) helps its own excision. Factor-assisted proofreading was shown to proceed through an exchange of active centres, a unique phenomenon among proteinaceous enzymes. Furthermore, most recent in vivo studies have revised the roles of transcription accuracy and proofreading factors, as not only required for production of errorless RNAs, but also for prevention of frequent misincorporation-induced pausing that may cause conflicts with fellow RNA polymerases and the replication machinery. [ABSTRACT FROM AUTHOR]

Some RNA classes require folding into the proper higher-order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA-binding ligand, naphthyridine carbamate tetramer with Z-stilbene linker ( Z-NCTS). Binding of Z-NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z-NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z-NCTS-responsive genetic switch using the HHR sequence inserted into the 3′ untranslated region as a cis-acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells. [ABSTRACT FROM AUTHOR]

Engineered ribozymes are powerful tools for manipulating gene expression in living cells. However, identification of active ribozymes in mammalian cells has relied on empirical assays of a limited number of arbitrarily chosen ribozymes. Here, we synthesized 376 natural and 2625 synthetic variants of pistol ribozymes, and screened for active variants directly in mammalian cells, greatly expanding the ribozyme toolbox for biological applications. [ABSTRACT FROM AUTHOR]

We have previously engineered new RNase P-based ribozyme variants with improved in vitro catalytic activity. In this study, we employed a novel engineered variant to target a shared mRNA region of human cytomegalovirus (HCMV) immediate early proteins 1 (IE1) and 2 (IE2), which are essential for the expression of viral early and late genes as well as viral growth. Ribozyme F-R228-IE represents a novel variant that possesses three unique base substitution point mutations at the catalytic domain of RNase P catalytic RNA. Compared to F-M1-IE that is the ribozyme derived from the wild type RNase P catalytic RNA sequence, the functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently. In cultured cells, expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98–99% and in HCMV production reduction by 50,000 folds. In contrast, expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds. Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression. Our results provide direct evidence that engineered RNase P ribozymes, such as F-R228-IE, can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection. Moreover, these results suggest that F-R228-IE, with novel and unique mutations at the catalytic domain to enhance ribozyme activity, can be a candidate for the construction of effective agents for anti-HCMV therapy. [ABSTRACT FROM AUTHOR]

RNA A-to-I editing is a post-transcriptional modification pervasively occurring in cells. Artificial intervention of A-to-I editing at specific sites of RNA could also be achieved with guide RNA and exogenous ADAR enzymes. In contrast to previous fused SNAP-ADAR enzymes for light-driven RNA A-to-I editing, we developed photo-caged antisense guide RNA oligonucleotides with simple 3′-terminal cholesterol modification, and successfully achieved light-triggered site-specific RNA A-to-I editing for the first time utilizing endogenous ADAR enzymes. Our caged A-to-I editing system effectively implemented light-dependent point mutation of mRNA transcripts of both exogenous and endogenous genes in living cells and 3D tumorspheres, as well as spatial regulation of EGFP expression, which provides a new approach for precise manipulation of RNA editing. [Display omitted] • Cholesterol conjugation may impair the function of oligonucleotides • Cholesterol-conjugated guide RNA is capable of photomanipulating RNA editing • Intracellular ADAR deaminases are employed in the photomanipulation of RNA editing Zhang et al. designed cholesterol-conjugated caged ADAR-recruiting antisense oligonucleotides through the temporary inhibition of their interaction with target RNAs, and the photomanipulation of site-specific RNA editing of target genes was successfully achieved at the presence of endogenous ADAR deaminases in both living cells and 3D tumorspheres. [ABSTRACT FROM AUTHOR]

RNAs assume sophisticated structures that are active in myriad cellular processes. In this review, we highlight newly identified ribozymes, riboswitches, and small RNAs, some of which control the function of cellular metabolic and gene expression networks. We then examine recent developments in genome-wide RNA structure probing technologies that are yielding new insights into the structural landscape of the transcriptome. Finally, we discuss how these RNA ‘structomic’ methods can address emerging questions in RNA systems biology, from the mechanisms behind long non-coding RNAs to new bases for human diseases. [ABSTRACT FROM AUTHOR]

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

In vitro compartmentalization (IVC) is a method to link genotype and phenotype by confining DNA and an in vitro gene expression system in cell-like compartments such as water-in-oil microdroplets. IVC provides a flexible platform for the selection and directed evolution of peptides, proteins, and RNAs with the desired catalytic, binding, and regulatory activities. However, in general, IVC requires confinement of a gene library in each compartment at the single-molecule level, resulting in the low expression level and activity of the protein and difficulty in recovering the DNA. To overcome the problems, we devised an alternative approach using microbeads displaying multiple copies of single genes prepared by on-bead emulsion PCR. We showed that this approach could increase the levels of protein synthesized compared with conventional IVC. Moreover, by employing this approach, we screened a library of green fluorescent protein (GFP) genes containing random sequences in the chromophore region and obtained genes encoding GFPs with different spectral characteristics in only a single round of screening. The result shows that our approach has great potential for practical applications in improving the properties or identifying new properties of enzymes, ribozymes, and their regulators. • An approach for a productive in vitro compartmentalization process is presented. • The approach uses microbeads displaying multiple copies of single genes. • The approach increased the levels of protein synthesized in microcompartments. • The approach enabled the acquisition of proteins with a desired property. [ABSTRACT FROM AUTHOR]