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Sidney Altmans 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.

Ribozymes, widely found in prokaryotes and eukaryotes, target nucleic acids and can be engineered as biotechnical tools or for gene regulation or immune therapy. Among them, hammerhead is the smallest and best characterized ribozyme. However, the structure and biochemical data of ribozymes have been disagreed on, making the understanding of its catalysis mechanism a longstanding issue. Particularly, the role of conformational dynamics in ribozyme catalysis remains elusive. Here, we use single-molecule magnetic tweezers to reveal a concerted catalysis mechanism of mechanical conformational selection for a mini hammerhead ribozyme against a viral RNA sequence from the SARS-CoV-2. We identify a conformational set containing five mechanical conformers of the mini ribozyme, where magnesium ions select the active one. Our results are supported by molecular dynamics simulations. Our understanding of the RNA catalytic mechanism will be beneficial for ribozyme's biotechnological applications and as potential therapeutics against RNA viruses. The catalysis of a ribozyme targeting the RNA sequence of SARS-CoV-2 involves a conformational selection mechanism as revealed by a single-molecule mechanical conformer probing method. [ABSTRACT FROM AUTHOR]

The formation of polymers that can hold gene information and work as catalysts is a crucial step for the origin of life. The discovery of catalytic RNA (i.e., ribozyme) supports the hypothesis that RNA might have served these functions at the early stage of life on the Earth. Given this, the spontaneous formation of RNA monomers (i.e., ribonucleotides) and their polymerization on Hadean Earth are essential steps for the origin of life. Previous experiments have investigated the chemical reactions that allow the formation of ribonucleotides and their components. These works have revealed the required molecules to form biological ribonucleotides (i.e., canonical ribonucleotides). Based on geochemical perspectives, abundantly available reactive molecules spontaneously react with each other to provide abundant products. Aldehydes and ammonia are reactive molecules assumed to have been present in considerable amounts on Hadean Earth. However, little is understood about whether or not nucleotides and their components were formed from these molecules under prebiotic conditions. We investigated the incubation products of alkaline aqueous solutions of aldehydes and ammonia. The product solution contained sugars (including ribose), various imidazole derivatives, and ribosyl imidazole (i.e., imidazole ribonucleoside). Ribosyl imidazole is formed via ribosyl amine, which reveals a new reaction pathway for prebiotic ribonucleoside synthesis. The imidazole ribonucleoside was then phosphorylated to imidazole ribonucleotide via a simple dry-down reaction with phosphate. Borate ion improved the reaction yields of these nucleosides and nucleotides. Because all the reactants were available on prebiotic Earth and the reactions progressed spontaneously, imidazole ribonucleotides could have accumulated in prebiotic environments. The experimental simplicity of the present reaction suggests that imidazoles were more abundant than canonical nucleobases on the prebiotic Earth. This further implies that prebiotic oligonucleotides contained imidazole bases in addition to the canonical nucleobases. The improvement of the reaction yields by borate indicates that borate-rich environments were conducive places for the formation and accumulation of non-canonical nucleosides and nucleotides. Such environments could have facilitated the formation of primordial ribonucleic acids on Hadean Earth. [ABSTRACT FROM AUTHOR]

Among the long non-coding RNAs that are currently recognized as important regulatory molecules influencing a plethora of processes in eukaryotic cells, circular RNAs (circRNAs) represent a distinct class of RNAs that are predominantly produced by back-splicing of pre-mRNA. The most studied regulatory mechanisms involving circRNAs are acting as miRNA sponges, forming R-loops with genomic DNA, and encoding functional proteins. In addition to circRNAs generated by back-splicing, two types of circRNAs capable of autonomous RNA-RNA replication and systemic transport have been described in plants: viroids, which are infectious RNAs that cause a number of plant diseases, and retrozymes, which are transcripts of retrotransposon genomic loci that are capable of circularization due to ribozymes. Based on a number of common features, viroids and retrozymes are considered to be evolutionarily related. Here, we provide an overview of the biogenesis mechanisms and regulatory functions of non-replicating circRNAs produced by back-splicing and further discuss in detail the currently available data on viroids and retrozymes, focusing on their structural features, replication mechanisms, interaction with cellular components, and transport in plants. In addition, biotechnological approaches involving replication-capable plant circRNAs are discussed, as well as their potential applications in research and agriculture. [ABSTRACT FROM AUTHOR]

Despite their importance in a wide range of living organisms, self-cleaving ribozymes in the human genome are few and poorly studied. Here, we performed deep mutational scanning and covariance analysis of two previously proposed self-cleaving ribozymes (LINE-1 and OR4K15). We found that the regions essential for ribozyme activities are made of two short segments, with a total of 35 and 31 nucleotides only. The discovery makes them the simplest known self-cleaving ribozymes. Moreover, the essential regions are circular permutated with two nearly identical catalytic internal loops, supported by two stems of different lengths. These two self-cleaving ribozymes, which are shaped like lanterns, are similar to the catalytic regions of the twister sister ribozymes in terms of sequence and secondary structure. However, the nucleotides at the cleavage site have shown that mutational effects on two twister sister-like (TS-like) ribozymes are different from the twister sister ribozyme. The discovery of TS-like ribozymes reveals a ribozyme class with the simplest and, perhaps, the most primitive structure needed for self-cleavage. [ABSTRACT FROM AUTHOR]

RNA interference, which enables more accurate downregulation of gene expression without interfering with the expression of various genes, is a beneficial instrument for gene silencing and a potential gene regulatory strategy in functional genomics that has a big impact on crop progress. Small interfering RNA molecules swift up the RNAi gene silencing machinery, effectually suppressing a target gene. Subsequently transgene proteins not present from genetically modified RNAi plants, it has a sum of rewards over orthodox transgenic methods. Dicer, Drosha which are RNA enzymes, along with protein complexes, RISC and Argonaute, institute at the epicenter of this technology. These explicit RNA binding proteins and ribonucleases regulate the synthesis and production of tiny regulating RNAs. Several latent usages for this ground-breaking process in agriculture have been identified for improving crops, such as the construction of disease-resistant, biotic or abiotic, high-yielding, stress-tolerant and exclusive assortments; enhancement of nutritional quality; removal of allergens and toxins and postponing the ripening of fruits and vegetables for extended storage periods. This ground-breaking procedure has the latent to be further utilized to pave the way for the creation of environmentally friendly biotech methods for crop protection and enhancement. Improving research and development activities in this auspicious field is imperative to ensure the efficient appropriate and safe application of these instruments as enduring remedies for agricultural enhancement. [ABSTRACT FROM AUTHOR]

Objective: Antisense oligonucleotide (ASO) and DNAzyme (Dz) agents have been suggested for suppression specific mRNA in vivo. It was reported that Dz agents are more selective in recognition their targets than ASO. However, Dz failed to produce therapeutically significant drugs due to their low efficiency. Here we compared the performance of the two types of agents in cleavage a folded RNA fragment in reconstituted system containing RNase H. Methods: Thermodynamic parameters and predicted 2D-structure of the RNA fragments were obtained using RNAFold application in UNAFold web server. To perform the experiments with RNA cleavage by enzymes we used commercial Mg2+-containing reaction 10X RNAse H Buffer (200 mM Tris-HCl (pH 8.3), 150 mM DTT, 1 M KCl, 45 mM MgCl2). Results of RNA cleavage were visualized with 20% denaturing PAGE (AA : BA (29 : 1), 7 M Urea, 1× TBE) running 150 min at 80 V. Results and Discussion: Individual ASO agents were ~3-–6 times more active in RNA cleaving than the equivalent Dz agents. Both agents demonstrated low selectivity toward RNA cleavage. Combining two Dz complementary to the abutting position of the RNA target bivalent (BDD) agent improved RNA cleavage to the level of the most active ASO agent. Conclusions: Comparing the obtained data with published earlier for RNase H—free system suggests that RNase H stabilizes the Dz:RNA complex and reduces its selectivity but significantly increase RNA cleavage efficiency. The contribution of RNase H effects on the performance of Dz agents in cell culture and in vivo should be taken in account. [ABSTRACT FROM AUTHOR]

Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation. [ABSTRACT FROM AUTHOR]

The prebiotic formation of RNA building blocks is well‐supported experimentally, yet the emergence of sequence‐ and structure‐specific RNA oligomers is generally attributed to biological selection via Darwinian evolution rather than prebiotic chemical selectivity. In this study, we used deep sequencing to investigate the partitioning of randomized RNA overhangs into ligated products by either splinted ligation or loop‐closing ligation. Comprehensive sequence‐reactivity profiles revealed that loop‐closing ligation preferentially yields hairpin structures with loop sequences UNNG, CNNG, and GNNA (where N represents A, C, G, or U) under competing conditions. In contrast, splinted ligation products tended to be GC rich. Notably, the overhang sequences that preferentially partition to loop‐closing ligation significantly overlap with the most common biological tetraloops, whereas the overhangs favoring splinted ligation exhibit an inverse correlation with biological tetraloops. Applying these sequence rules enables the high‐efficiency assembly of functional ribozymes from short RNAs without template inhibition. Our findings suggest that the RNA tetraloop structures that are common in biology may have been predisposed and prevalent in the prebiotic pool of RNAs, prior to the advent of Darwinian evolution. We suggest that the one‐step prebiotic chemical process of loop‐closing ligation could have favored the emergence of the first RNA functions. [ABSTRACT FROM AUTHOR]

Ribozymes are small catalytic RNA sequences capable of nucleotide-specific self-cleavage found widespread in nature. Ribozyme cleavage generates distinct 2′,3′-phosphate and 5′-hydroxyl termini that resemble substrates for recently characterized RNA repair pathways in cells. We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for Stitch RNA). Optimization of StitchR activity in mammalian cells resulted in a ~900-fold increase in protein expression that approached levels observed for genes expressed from single vectors. We demonstrate that StitchR can be harnessed for effective dual adeno-associated virus gene therapies to correct muscular dystrophies by restoring large functional muscle proteins to endogenous levels in vivo. [ABSTRACT FROM AUTHOR]

Summary: The functional relevance of plant‐associated microorganisms is theoretically framed within the holobiont concept. The role of viruses in plant holobionts is being recognized both for their direct effects when hosted in plants (cryptic plant viruses) and for their indirect effects when infecting microorganisms associated with plants in tripartite interactions (e.g. mycoviruses and bacteriophages). We argue that viroids, the smallest infectious agents typically infecting only plant hosts, must also be included in plant holobiont studies. The same applies to the recently discovered large number of viroid‐like elements infecting hosts of other life kingdoms that are closely associated with plants. Here we also describe in depth the diversity of such viroid‐like elements and their initial functional characterization in plant‐associated fungi. [ABSTRACT FROM AUTHOR]

RNA oxidation, predominantly through the accumulation of 8-oxo- 7,8- dihydroguanosine (8-oxo- rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo- rG- containing RNA and protect Escherichia coli cells from oxidative stress. However, the impact of 8-oxo- rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo- rG in RNA leads to catalytic stalling of E. coli PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo- rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo- rG- specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo- rG binding through E. coli cell growth experiments under H2O2-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo- rG- specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress. [ABSTRACT FROM AUTHOR]

Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) by ADAR1 is an essential modifier of the immunogenicity of cellular dsRNA. The role of MDA5 in sensing unedited cellular dsRNA and the downstream activation of type I interferon (IFN) signaling are well established. However, we have an incomplete understanding of pathways that modify the response to unedited dsRNA. We performed a genome-wide CRISPR screen and showed that GGNBP2, CNOT10, and CNOT11 interact and regulate sensing of unedited cellular dsRNA. We found that GGNBP2 acts between dsRNA transcription and its cytoplasmic sensing by MDA5. GGNBP2 loss prevented induction of type I IFN and autoinflammation after the loss of ADAR1 editing activity by modifying the subcellular distribution of endogenous A-to-I editing substrates and reducing cytoplasmic dsRNA load. These findings reveal previously undescribed pathways to modify diseases associated with ADAR mutations and may be determinants of response or resistance to small-molecule ADAR1 inhibitors. Editor's summary: Innate immune sensing of endogenous double-stranded RNA (dsRNA) is prevented by the A-to-I RNA editing enzyme ADAR1, mutations in which cause the autoinflammatory disease Aicardi-Goutières syndrome. Using a genome-wide screen in myeloid cells expressing an editing-dead ADAR1 protein, Heraud-Farlow et al. identified GGNBP2, CNOT10, and CNOT11 as key regulators of the autoinflammatory response to unedited self dsRNA. These proteins directly interacted, forming a GGNBP2-CNOT10/11 complex required for cytoplasmic accumulation of unedited dsRNA and sensing by MDA5. Together, these findings identify a previously undescribed pathway facilitating the cellular response to immunogenic self dsRNA. —Claire Olingy [ABSTRACT FROM AUTHOR]

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D-and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D-nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D-and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification. [ABSTRACT FROM AUTHOR]

The article focuses on the promising potential of RNA editing as a therapeutic approach for genetic disorders, highlighted through the story of a mother advocating for treatment options for her daughter with Rett syndrome. Topics include the historical context of Rett syndrome and its genetic basis, the current advancements and research funding in RNA editing therapies, and the advantages of RNA editing over traditional DNA-based therapies, including safety and temporary effects.

AbstractVarkud satellite ribozyme (VS ribozyme) is a class of catalytic RNA with self-cleavage activity. The wild-type VS ribozyme has structural modularity with a relatively large catalytic module (H2–H6 elements) and a small substrate module (H1 element). The two modules can be dissected physically, and the substrate H1 RNA is recognized and then cleaved by the rest of the parent ribozyme serving as catalytic RNA. We characterized the catalytic properties of a bimolecular VS ribozyme developed and employed for an in-droplet evolution experiment of the VS ribozyme. We examined the effects of polyamines and several divalent metal ions. The results obtained in this study would be useful for the optimization of laboratory evolution of the VS ribozyme. [ABSTRACT FROM AUTHOR]

The CRISPR‐Cas systems are adopted as powerful molecular tools for not only genetic manipulation but also point‐of‐care diagnostics. However, methods to enable diagnostics of non‐nucleic‐acid targets with these systems are still limited. Herein, by fusing ligand‐dependent allosteric ribozymes with CRISPR‐Cas12a, a derived CRISPR‐Cas system is created for efficient quantitative analysis of non‐nucleic‐acid targets in 1–2 h. On two different small molecules, the system's generality, reliability and accuracy is demonstrated, and show that the well operability of this system can enable high‐throughput detection of a small molecule in blood samples. The system can be further converted to rely on allosteric deoxyribozyme instead of allosteric ribozyme to recognize non‐nucleic‐acid targets and transduce the signal to CRISPR‐Cas12a for amplification, likely making it easier for storage and more consistent in data generation as DNA possess a stability advantage over RNA. This (deoxy)ribozyme‐assisted CRISPR‐Cas12a system anticipates that it can facilitate bioanalysis in various scientific and clinical settings and further drive the development of clinical translation. [ABSTRACT FROM AUTHOR]

Our research elucidates the cleavage processes of the RNase III enzyme, DICER, which plays a crucial role in the production of small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Utilizing high-throughput dicing assays, we expose the bipartite pairing rule that dictates the cleavage sites of DICER. Furthermore, we decode the intricate recognition mechanism of the primary YCR motif and identify an analogous secondary YCR motif that influences DICER's cleavage choices. Collectively, our findings clarify the bipartite pairing rule and enhance our understanding of the role of RNA motifs in modulating DICER's cleavage activity, laying the groundwork for future research on their roles in miRNA biogenesis and gene regulation. DICER is a vital enzyme in small RNA production. Here the authors show how bipartite pairing rules and dual YCR motifs guide DICER's activity on precursor microRNAs. [ABSTRACT FROM AUTHOR]

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5′ direction (5′-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5′-G (−1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5′-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5′-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the −1 position significantly improves editing at 5′-GA sites. [ABSTRACT FROM AUTHOR]

Group I and II introns are large catalytic RNAs (ribozymes) that are frequently encountered in fungal mitochondrial genomes. The discovery of respiratory mutants linked to intron splicing defects demonstrated that for the efficient removal of organellar introns there appears to be a requirement of protein splicing factors. These splicing factors can be intron-encoded proteins with maturase activities that usually promote the splicing of the introns that encode them (cis-acting) and/or nuclear-encoded factors that can promote the splicing of a range of different introns (trans-acting). Compared to plants organellar introns, fungal mitochondrial intron splicing is still poorly explored, especially in terms of the synergy of nuclear factors with intron-encoded maturases that has direct impact on splicing through their association with intron RNA. In addition, nuclear-encoded accessory factors might drive the splicing impetus through translational activation, mitoribosome assembly, and phosphorylation-mediated RNA turnover. This review explores protein-assisted splicing of introns by nuclear and mitochondrial-encoded maturases as a means of mitonuclear interplay that could respond to environmental and developmental factors promoting phenotypic adaptation and potentially speciation. It also highlights key evolutionary events that have led to changes in structure and ATP-dependence to accommodate the dual functionality of nuclear and organellar splicing factors. [ABSTRACT FROM AUTHOR]

BACKGROUND: Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap‐sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV‐tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. RESULTS: Insecticide susceptible D. citri reared on citrus infected with CTV‐tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non‐infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV‐tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field‐collected psyllids from areas with known broad‐spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV‐tCYP450 citrus trees as compared with those reared on controls. CONCLUSION: The integration of citrus‐mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis. [ABSTRACT FROM AUTHOR]

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis. [ABSTRACT FROM AUTHOR]

tRNAs have recently gained attention for their novel regulatory roles in translation and for their diverse functions beyond translation. One of the most remarkable aspects of tRNA biogenesis is the incorporation of various chemical modifications, ranging from simple base or ribose methylation to more complex hypermodifications such as formation of queuosine and wybutosine. Some tRNAs are transcribed as intron-containing pre-tRNAs. While the majority of these modifications occur independently of introns, some are catalyzed in an intron-inhibitory manner, and in certain cases, they occur in an intron-dependent manner. This review focuses on pre-tRNA modification, including introncontaining pre-tRNA, in both intron-inhibitory and intron-dependent fashions. Any perturbations in the modification and processing of tRNAs may lead to a range of diseases and disorders, highlighting the importance of understanding these mechanisms in molecular biology and medicine. [ABSTRACT FROM AUTHOR]

Modern life is essentially homochiral, containing D-sugars in nucleic acid backbones and L-amino acids in proteins. Since coded proteins are theorized to have developed from a prebiotic RNA World, the homochirality of L-amino acids observed in all known life presumably resulted from chiral transfer from a homochiral D-RNA World. This transfer would have been mediated by aminoacyl-RNAs defining the genetic code. Previous work on aminoacyl transfer using tRNA mimics has suggested that aminoacylation using D-RNA may be inherently biased toward reactivity with L-amino acids, implying a deterministic path from a D-RNA World to L-proteins. Using a model system of self-aminoacylating D-ribozymes and epimerizable activated amino acid analogs, we test the chiral selectivity of 15 ribozymes derived from an exhaustive search of sequence space. All of the ribozymes exhibit detectable selectivity, and a substantial fraction react preferentially to produce the D-enantiomer of the product. Furthermore, chiral preference is conserved within sequence families. These results are consistent with the transfer of chiral information from RNA to proteins but do not support an intrinsic bias of D-RNA for L-amino acids. Different aminoacylation structures result in different directions of chiral selectivity, such that L-proteins need not emerge from a D-RNA World. Life is homochiral, and prior work posited that D-RNA may interact preferentially with L-amino acids. Here, the authors show that self-aminoacylating D-RNAs do not have an intrinsic preference for L-amino acids, suggesting that either handedness could emerge from early living systems. [ABSTRACT FROM AUTHOR]

Proteins are synthesized within ribosomes through the polymerization of amino acids (AAs). This process requires prior activation of AAs through aminoacylation that attaches them to their corresponding transfer RNAs (tRNAs). Within cells, this attachment is facilitated by aminoacyl‐tRNA synthetase, resulting in a tRNA:AA conjugate. A set of ribozymes developed to acylate tRNA with non‐canonical substrates enables this process outside the confines of living cells, thereby facilitating the synthesis of novel bio‐based products. In modern biotechnology, aminoacylating ribozymes contribute to the production of innovative bio‐based materials bearing functional non‐canonical chemical substrates (NCSs) and fill the gaps in synthesizing unique polymeric backbones, extending the scope beyond traditional peptide bonds. This review summarizes current understanding of flexizymes at the molecular level and their application in generating exceptional polymeric backbones through ribosome‐mediated synthesis in vitro. [ABSTRACT FROM AUTHOR]

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]

Following the success of mRNA vaccines against COVID-19, mRNA-based therapeutics have now become a great interest and potential. The development of this approach has been preceded by studies of modifications found on mRNA ribonucleotides that influence the stability, translation and immunogenicity of this molecule. The 5′ cap of eukaryotic mRNA plays a critical role in these cellular functions and is thus the focus of intensive chemical modifications to affect the biological properties of in vitro-prepared mRNA. Enzymatic removal of the 5′ cap affects the stability of mRNA in vivo. The NUDIX hydrolase Dcp2 was identified as the first eukaryotic decapping enzyme and is routinely used to analyse the synthetic cap at the 5′ end of RNA. Here we highlight three additional NUDIX enzymes with known decapping activity, namely Nudt2, Nudt12 and Nudt16. These enzymes possess a different and some overlapping activity towards numerous 5′ RNA cap structures, including non-canonical and chemically modified ones. Therefore, they appear as potent tools for comprehensive in vitro characterisation of capped RNA transcripts, with special focus on synthetic RNAs with therapeutic activity. [ABSTRACT FROM AUTHOR]

In vitro circular RNA (circRNA) preparation methods have been gaining a lot of attention recently as several reports suggest that circRNAs are more stable, with better performances in cells and in vivo, than linear RNAs in various biomedical applications. Self-splicing ribozymes are considered a major in vitro circRNA generation method for biomedical applications due to their simplicity and efficiency in the circularization of the gene of interest. This review summarizes, updates, and discusses the recently developed self-circularization methods based on the self-splicing ribozyme, such as group I and II intron ribozymes, and the pros and cons of each method in preparing circRNA in vitro. [ABSTRACT FROM AUTHOR]

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1–244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes. SICKLE sequestrates DBR1 into stress granules to promote the accumulation of lariRNAs, which upregulate heat stress-related genes and enhance heat-stress tolerance. [ABSTRACT FROM AUTHOR]

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis. [ABSTRACT FROM AUTHOR]

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp–Glu–Ala–Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21- sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation. [ABSTRACT FROM AUTHOR]

Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments. [ABSTRACT FROM AUTHOR]

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR. [ABSTRACT FROM AUTHOR]

Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5′->3′ exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal. [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]

Nucleic acid-based gene interference and editing strategies, such as antisense oligonucleotides, ribozymes, RNA interference (RNAi), and CRISPR/Cas9 coupled with guide RNAs, are exciting research tools and show great promise for clinical applications in treating various illnesses. RNase P ribozymes have been engineered for therapeutic applications against human viruses such as human cytomegalovirus (HCMV). M1 ribozyme, the catalytic RNA subunit of RNase P from Escherichia coli, can be converted into a sequence-specific endonuclease, M1GS ribozyme, which is capable of hydrolyzing an mRNA target base-pairing with the guide sequence. M1GS RNAs have been shown to hydrolyze essential HCMV mRNAs and block viral progeny production in virus-infected cell cultures. Furthermore, RNase P ribozyme variants with enhanced hydrolyzing activity can be generated by employing in vitro selection procedures and exhibit better ability in suppressing HCMV gene expression and replication in cultured cells. Additional studies have also examined the antiviral activity of RNase P ribozymes in mice in vivo. Using cytomegalovirus infection as an example, this review summarizes the principles underlying RNase P ribozyme-mediated gene inactivation, presents recent progress in engineering RNase P ribozymes for applications in vitro and in mice, and discusses the prospects of using M1GS technology for therapeutic applications against HCMV as well as other pathogenic viruses. [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]

Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to also play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease. [ABSTRACT FROM AUTHOR]

We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozyme that closely resembles the pistol ribozyme, with essentially identical secondary structure and connectivity. The activity is more sensitive to deletion of the G8 2'OH than to the absence of magnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead, and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line, and the G8 2'OH is well positioned to act as general acid, being directed towards the O5' leaving group, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, the local structure close to the cleavage site differs, and the PK hammerhead retains its unique mechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes under standard conditions. Here the authors report a crystal structure of a pseudoknot-containing hammerhead ribozyme. Catalysis is based upon a general base (G N1) and a general acid (G O2'). The structure is closely similar to the pistol ribozyme, but mechanistically different. [ABSTRACT FROM AUTHOR]

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented. [ABSTRACT FROM AUTHOR]

SUMMARY: The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We assembled the P. drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events. In addition to many C‐to‐U editing events in both organelles, we found just four U‐to‐C editing events in the mitochondrial transcripts cob, nad1, nad5 and rpl2. These events are conserved in related lycophytes in the genera Huperzia and Phlegmariurus. De novo transcriptomes for three of these lycophytes were assembled to search for putative U‐to‐C RNA editing enzymes. Four putative U‐to‐C editing factors could be matched to the four mitochondrial U‐to‐C editing sites. Due to the unusually few numbers of U‐to‐C RNA editing sites, P. drummondii and related lycophytes are useful models for studying this poorly understood mechanism. [ABSTRACT FROM AUTHOR]

RNA profiling studies have revealed that ∼75% of the human genome is transcribed to RNA but only a meagre fraction of it is translated to proteins. Majority of transcribed RNA constitute a specialized pool of non-coding RNAs. Human genome contains approximately 506 genes encoding a set of 51 different tRNAs, constituting a unique class of non-coding RNAs that not only have essential housekeeping functions as translator molecules during protein synthesis, but have numerous uncharted regulatory functions. Intriguing findings regarding a variety of non-canonical functions of tRNAs, tRNA derived fragments (tRFs), esoteric epitranscriptomic modifications of tRNAs, along with aminoacyl-tRNA synthetases (AARSs) and ARS-interacting multifunctional proteins (AIMPs), envision a 'peripheral dogma' controlling the flow of genetic information in the backdrop of qualitative information wrung out of the long-live central dogma of molecular biology, to drive cells towards either proliferation or differentiation programs. Our review will substantiate intriguing peculiarities of tRNA gene clusters, atypical tRNA-transcription from internal promoters catalysed by another distinct RNA polymerase enzyme, dynamically diverse tRNA epitranscriptome, intricate mechanism of tRNA-charging by AARSs governing translation fidelity, epigenetic regulation of gene expression by tRNA fragments, and the role of tRNAs and tRNA derived/associated molecules as quantitative determinants of the functional proteome, covertly orchestrating the process of tumorigenesis, through a deregulated tRNA-ome mediating selective codon-biased translation of cancer related gene transcripts. [Display omitted] • Distinct tRNA repertoire undertakes codon-biased translation of cancer linked genes. • tRNA deregulation & mischarging diversify tumor proteome entailing chemo-resistance. • Deregulated 'writers' alter the epitranscriptome & stabilize cancerous tRNA profile. • As part of MSC, AARS-AIMPs play non-canonical oncogenic/tumor-suppressive roles. • tRFs/tRDs fine-tune epigenetic regulation of genes involved in tumour progression. [ABSTRACT FROM AUTHOR]

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as ‘ambiviruses,’ were described. ‘Ambiviruses’ have single-stranded RNA genomes of about 4–5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known ‘ambiviruses,’ and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, ‘ambivirus’ genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations—features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as ‘epsilonviruses,’ ‘zetaviruses,’ and some ‘obelisks’). As a first step toward the formal classification of ‘ambiviruses,’ the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences. [ABSTRACT FROM AUTHOR]

Observational and experimental discoveries of new factual entities such as objects, systems, or processes, are major contributors to some advances in the life sciences. Yet, whereas discovery of theories was extensively deliberated by philosophers of science, very little philosophical attention was paid to the discovery of factual entities. This paper examines historical and philosophical aspects of the experimental discovery by Carl Woese of archaea, prokaryotes that comprise one of the three principal domains of the phylogenetic tree. Borrowing Kuhn's terminology, this discovery of a major biological entity was made during a 'normal science' project of building molecular taxonomy for prokaryotes. Unexpectedly, however, an observed anomaly instigated the discovery of archaea. Substantiation of the existence of the new archaeal entity and consequent reconstruction of the phylogenetic tree prompted replacement of a long-held model of a prokarya and eukarya bipartite tree of life by a new model of a tripartite tree comprising of bacteria, archaea, and eukarya. This paper explores the history and philosophical implications of the progression of Woese's project from normal science to anomaly-instigated model-changing discovery. It is also shown that the consequential discoveries of RNA splicing and of ribozymes were similarly prompted by unexpected irregularities during normal science activities. It is thus submitted that some discoveries of factual biological entities are triggered by unforeseen observational or experimental anomalies. [ABSTRACT FROM AUTHOR]

Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures. [ABSTRACT FROM AUTHOR]

This special issue of JBC pays tribute to Sidney Altman, whose discovery of a catalytic role for RNA, a breakthrough made independently by Thomas Cech, overturned the longheld dogma that only proteins can serve as catalysts in biological systems. The discovery of RNA catalysis galvanized biologists to think expansively in new directions and has given rise to a remarkable RNAissance in science and medicine. The collection of articles begins with the story of the discovery of RNase P and builds up to the emerging picture of an unexpectedly vast repertoire of RNase P variants in the three domains of life, including insights derived from recent highresolution structures on how RNAs, ribonucleoproteins, or protein scaffolds can be used variably to generate an active site for catalyzing the same RNA processing reaction. The series of articles ends with a discussion of more recently discovered endonucleases (Argonautes, Cas), whose parallels with RNase P underscore recurring themes in diverse biological contexts. [ABSTRACT FROM AUTHOR]