RNA World Research Articles

A Plausible Prebiotic Path to Nucleosides: Ribosides and Related Aldosides Generated from Ribulose, Fructose, and Similar Abiotic Precursors.

The prebiotic origins of ribose, nucleosides, and eventually RNA are enduring questions whose answers are central to the RNA world hypothesis. The abiotic synthesis of sugars was first demonstrated over a century ago, but no known prebiotic reaction produces ribose (an aldose sugar) selectively and in good yield. In contrast, ribulose, and fructose (ketose sugars) and other monosaccharides are formed in high yield by several robust abiotic reactions. It is reported here that ketose sugars - both ketopentoses and ketohexoes - serve as precursors for the formation of ribosides and other aldosides, as demonstrated by glycoside-forming reactions involving barbituric acid, a plausibly prebiotic nucleobase. Moreover, a one-pot reaction of glyceraldehyde and barbituric acid was discovered which under mild conditions, and without special minerals or other catalysts, results in the formation of glycosides. These results reveal that an exclusive or high-yielding generation of free ribose was not required for its incorporation into processes that provided the foundations for life.

A large-scale benchmark study of tools for the classification of protein-coding and non-coding RNAs.

Identification of protein-coding and non-coding transcripts is paramount for understanding their biological roles. Computational approaches have been addressing this task for over a decade; however, generalized and high-performance models are still unreliable. This benchmark study assessed the performance of 24 tools producing >55 models on the datasets covering a wide range of species. We have collected 135 small and large transcriptomic datasets from existing studies for comparison and identified the potential bottlenecks hampering the performance of current tools. The key insights of this study include lack of standardized training sets, reliance on homogeneous training data, gradual changes in annotated data, lack of augmentation with homology searches, the presence of false positives and negatives in datasets and the lower performance of end-to-end deep learning models. We also derived a new dataset, RNAChallenge, from the benchmark considering hard instances that may include potential false alarms. The best and least well performing models under- and overfit the dataset, respectively, thereby serving a dual purpose. For computational approaches, it will be valuable to develop accurate and unbiased models. The identification of false alarms will be of interest for genome annotators, and experimental study of hard RNAs will help to untangle the complexity of the RNA world.

Ancestral Sequence Reconstruction of the Ribosomal Protein uS8 and Reduction of Amino Acid Usage to a Smaller Alphabet.

Understanding the origin and early evolution of proteins is important for unveiling how the RNA world developed into an RNA-protein world. Because the composition of organic molecules in the Earth's primitive environment was plausibly not as diverse as today, the number of different amino acids used in early protein synthesis is likely to be substantially less than the current 20 proteinogenic residues. In this study, we have explored the thermal stability and RNA binding of ancestral variants of the ribosomal protein uS8 constructed from a reduced-alphabet of amino acids. First, we built a phylogenetic tree based on the amino acid sequences of uS8 from multiple extant organisms and used the tree to infer two plausible amino acid sequences corresponding to the last bacterial common ancestor of uS8. Both ancestral proteins were thermally stable and bound to an RNA fragment. By eliminating individual amino acid letters and monitoring thermal stability and RNA binding in the resulting proteins, we reduced the size of the amino acid set constituting one of the ancestral proteins, eventually finding that convergent sequences consisting of 15- or 14-amino acid alphabets still folded into stable structures that bound to the RNA fragment. Furthermore, a simplified variant reconstructed from a 13-amino-acid alphabet retained affinity for the RNA fragment, although it lost conformational stability. Collectively, RNA-binding activity may be achieved with a subset of the current 20 amino acids, raising the possibility of a simpler composition of RNA-binding proteins in the earliest stage of protein evolution.

Abiogenesis through gradual evolution of autocatalysis into template-based replication.

How life emerged from inanimate matter is one of the most intriguing questions posed to modern science. Central to this research are experimental attempts to build systems capable of Darwinian evolution. RNA catalysts (ribozymes) are a promising avenue, in line with the RNA world hypothesis whereby RNA pre-dated DNA and proteins. Since evolution in living organisms relies on template-based replication, the identification of a ribozyme capable of replicating itself (an RNA self-replicase) has been a major objective. However, no self-replicase has been identified to date. Alternatively, autocatalytic systems involving multiple RNA species capable of ligation and recombination may enable self-reproduction. However, it remains unclear how evolution could emerge in autocatalytic systems. In this review, we examine how experimentally feasible RNA reactions catalysed by ribozymes could implement the evolutionary properties of variation, heredity and reproduction, and ultimately allow for Darwinian evolution. We propose a gradual path for the emergence of evolution, initially supported by autocatalytic systems leading to the later appearance of RNA replicases.

Christine Guthrie: Splicing genetics and mentorship into the RNA world

Christine Guthrie: Splicing genetics and mentorship into the RNA world

Discovering glycoRNA: Traditional and Non‐Canonical Approaches to Studying RNA Modifications

AbstractChemical biology opens the door to understanding biological processes in many areas, but nowhere has it had a greater impact on defining a field than in glycobiology. The Wolf and Nobel Prize‐winning work by Carolyn Bertozzi and coworkers to conceptualize bioorthogonal chemistry, enable it on living cells, deploy it with metabolic chemical reporters (MCRs) such as N‐azidoacetylmannosamine‐tetraacylated (Ac4ManNAz), has allowed researchers to elucidate the structure, functions, and regulation of complex glycans across various domains of life. In this review, we highlight how our work using Ac4ManNAz to discover glycosylated RNA (glycoRNA) shed the traditional paradigms of RNA post‐transcriptional modification discovery. While our understanding of RNA modifications has historically been expanded with unbiased chemical tools, the creative use of Ac4ManNAz for the targeted study of RNA's intersection with glycobiology has advanced Bertozzi's impact into the world of RNA.

Navigating the Multiverse of Antisense RNAs: The Transcription- and RNA-Dependent Dimension.

Evidence accumulated over the past decades shows that the number of identified antisense transcripts is continuously increasing, promoting them from transcriptional noise to real genes with specific functions. Indeed, recent studies have begun to unravel the complexity of the antisense RNA (asRNA) world, starting from the multidimensional mechanisms that they can exert in physiological and pathological conditions. In this review, we discuss the multiverse of the molecular functions of asRNAs, describing their action through transcription-dependent and RNA-dependent mechanisms. Then, we report the workflow and methodologies to study and functionally characterize single asRNA candidates.

Pressure-temperature control of activity of RNA polymerase ribozyme

A representative role of nucleic acids (DNA and RNA) is in the storage of genetic information. In contrast, RNAs act as ribozymes that catalyze various biochemical reactions. The “RNA world” hypothesis suggests that the origin of life was RNA because a ribozyme that shows RNA replication activity has been identified. However, prebiotic conditions in the RNA world remain unknown. In this study, we investigated the effect of high pressure and temperature on RNA replication using an RNA polymerase ribozyme tC9Y. We found that pressure accelerated the RNA replication activity of tC9Y ribozyme at higher temperatures than physiological conditions. Furthermore, molecular crowding by concentrated polyethylene glycol 200 (average molecular weight 200) synergistically enhanced the replication activity at higher pressure and temperature because the negative effect of a volumetric contribution of hydration on the tC9Y ribozyme activity decreased under crowding conditions. As a comparison, proteinaceous RNA polymerase that exists in the modern era did not show accelerated activity under high pressure and temperature. Thus, these results imply that the prebiotic conditions for the RNA world were at high pressure and temperatures under crowding conditions.

Brain Metabolic DNA: A Long Story and Some Conclusions.

We have previously outlined the main properties of brain metabolic DNA (BMD) and its involvement in circadian oscillations, learning, and post-trial sleep. The presence of BMD in certain subcellular fractions and their behavior in cesium gradients have suggested that BMD originates from cytoplasmic reverse transcription and subsequently acquires a double-stranded configuration. More recently, it has been reported that some DNA sequences of cytoplasmic BMD in learning mice are different from that of the control animals. Furthermore, BMD is located in vicinity of the genes involved in different modifications of synaptic activity, suggesting that BMD may contribute to the brain's response to the changing environment. The present review outlines recent data with a special emphasis on reverse transcription of BMD that may recapitulate the molecular events at the time of the "RNA world" by activating mitochondrial telomerase and generating RNA templates from mitochondrial transcripts. The latter unexpected role of mitochondria is likely to promote a better understanding of mitochondrial contribution to cellular interactions and eukaryotic evolution. An initial step regards the role of human mitochondria in embryonic BMD synthesis, which is exclusively of maternal origin. In addition, mitochondrial transcripts involved in reverse transcription of BMD might possibly reveal unexpected features elucidating mitochondrial involvement in cancer events and neurodegenerative disorders.

News from around the RNA world: new avenues in RNA biology, biotechnology and therapeutics from the 2022 SIBBM meeting.

ABSTRACTSince the formalization of the Central Dogma of molecular biology, the relevance of RNA in modulating the flow of information from DNA to proteins has been clear. More recently, the discovery of a vast set of non-coding transcripts involved in crucial aspects of cellular biology has renewed the enthusiasm of the RNA community. Moreover, the remarkable impact of RNA therapies in facing the COVID19 pandemics has bolstered interest in the translational opportunities provided by this incredible molecule. For all these reasons, the Italian Society of Biophysics and Molecular Biology (SIBBM) decided to dedicate its 17th yearly meeting, held in June 2022 in Rome, to the many fascinating aspects of RNA biology. More than thirty national and international speakers covered the properties, modes of action and applications of RNA, from its role in the control of development and cell differentiation to its involvement in disease. Here, we summarize the scientific content of the conference, highlighting the take-home message of each presentation, and we stress the directions the community is currently exploring to push forward our comprehension of the RNA World 3.0.

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of “multiple” functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

Molecular Mechanisms of Phosphoester Bond Formation in Water Using Tight-Binding Ab Initio Molecular Dynamics.

Phosphate groups are ubiquitous in biomolecules and are usually incorporated through phosphoester bonds between alcohol groups and orthophosphate. The formation of this bond is exceptionally difficult, with associated barriers of 30-45 kcal/mol in the absence of catalysts. In abiotic conditions, polymerizing nucleic acids without enzymes remains very challenging and is still a partly unsolved problem that severely questions the RNA World hypothesis for the origins of life. Offering a solution to this problem would involve a detailed knowledge of the reaction energetics and mechanisms, yet these remain not fully understood at a molecular level, especially because of the very slow reaction rates that represent a significant challenge for the experiments. The number of involved reaction coordinates and the possible role of the solvent in assisting the reaction are challenging for computational studies. Here, we use extensive ab initio molecular dynamics simulations using semiempirical tight-binding methods and enhanced sampling to address these issues. We first show that the choice of the tight-binding method is greatly limited by the instability of the water liquid phase for most DFTB generations and parameter sets that are widely available. We then focus on a model reaction involving methanol and orthophosphate, for which the two protonation states (mono- and dianionic) that are dominant around neutral pH are considered. We compare different proton coordinates that enable (or not) the participation of solvent water molecules. Our simulations suggest that in all cases, a dissociative associative mechanism, with an intermediate metaphosphate, is favored. The main difference between the two phosphate species is that reaction with the monoanion is assisted by the substrate, while that with the dianion involves solvent water molecules. Our results are in agreement with early experimental measurements, but the reaction barriers are underestimated in our framework. We believe that our approach provides an interesting perspective on how to sample the reaction phase space efficiently, but it calls for future studies using more accurate descriptions of chemical reactivity.

An RNA aptamer that shifts the reduction potential of metabolic cofactors

The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we develop single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and helps re-activate RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

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

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

A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme.

The RNA world hypothesis suggests that chemical networks consisting of functional RNA molecules could have constructed a primitive life-like system leading a first living system. The chemical evolution scenario of RNA molecules should be consistent with the Hadean Earth environment. We have demonstrated the importance of the environment at both high temperature and high pressure, using different types of hydrothermal flow reactor systems and high-pressure equipment. In the present study, we have attempted to develop an alternative easy-to-implement method for high-pressure measurements and demonstrate that the system is applicable as an efficient research tool for high-pressure experiments at pressures up to 30 MPa. We demonstrate the usefulness of the system by detecting the high-pressure influence for the self-cleavage of avocado hammerhead ribozyme (ASBVd(−):HHR) at 45–65 °C. A kinetic analysis of the high-pressure behavior of ASBVd(−):HHR shows that the ribozyme is active at 30 MPa and its activity is sensitive to pressures between 0.1–30 MPa. The surprising finding that such a short ribozyme is effective for self-cleavage at a high pressure suggests the importance of pressure as a factor for selection of adaptable RNA molecules towards an RNA-based life-like system in the Hadean Earth environment deep in the ocean.

Editorial: RNA world hypothesis and the origin of life: Astrochemistry perspective

Editorial: RNA world hypothesis and the origin of life: Astrochemistry perspective

Methanol in the RNA world: An astrochemical perspective

The role and relevance of methanol in the origin and structure of the RNA world is discussed. Methanol is a pivotal, renewable, and regenerable source from which almost all chemical materials, simple or complex, can be accessed. Olefins and carbonyl compounds, amines and amino acids, peptides and polypeptides, and the molecular building blocks in the initial stages of the biological evolution to life’s origin are obtained through methanol as a source material by its chemical transformation. The formation of methanol, whether in stellar and interstellar media, in deep sea-bottom hot hydrothermal vents or from geothermal sources, results from CO2 hydrogenation. It is the basic reaction, setting the stage for the formation of fundamental “organic” building blocks for the formation of simple prebiotic cells to subsequent biological evolution to cells. The important observation of many organics– hydrocarbons and ions including the large expanse of methane and methanol in the interstellar medium and stellar peripheries is a clear indication of “stellar reductive processes” and ensuing reactions shedding light on the probable significant role of extraterrestrial methanol as the basic source material toward a multi-step transformation into complex life molecules such as RNA.

Entering the labyrinth: A hypothesis about the emergence of metabolism from protobiotic routes

The occurrence of organized chemical transformations defined as metabolism is one of the most important characteristics of life. Surprisingly though, there is not a consensus about how those transformations were originated in the origin of life. RNA world advocates suggest that biochemical pathways started with ribozymes that were further substituted by enzymes. However, most of the biosynthetic routes of ribozymes described do not overlap with the enzymatic routes, and there is not a clear theory about how this transition happened. An important step to solve this dilemma has been elucidated in the last decade when researchers found that some complex routes of chemical transformations, such as the glycolytic and the citric acid pathways, already existed in prebiotic Earth due to physicochemical forces alone. Defined here as protobiotic pathways, we propose that those metabolic exchanges working without the aiding of any biological catalysts were the ones that guided the origin of metabolism. Under this scenario, some quasi-randomly encoded peptides at the origins of translation systems would be capable to bind metabolites in protobiotic routes. When those bounds facilitated or accelerated the conversion of metabolites along the protobiotic path and the products were beneficial, then natural molecular selection acted to preserve the system. Thus, we propose that the origin of metabolism happened when peptides started to bind metabolites in protobiotic routes without disturbing (and possibly aiding) their chemical transformation paths. This should have been the entry point to the metabolic labyrinth, the key step that allowed peptides to come into the path of chemical transformations and further evolve into the enzymes that coordinate nowadays the biochemical pathways.

Phylogenic analysis of new viral cluster of large phages with unusual DNA genomes containing uracil in place of thymine in gene-sharing network, using phages S6 and PBS1 and relevant uncultured phages derived from sewage metagenomics

Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed “dU jumbophages” in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria.