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Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today's nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the "water problem" that prohibits the synthesis of nucleotides in water. [ABSTRACT FROM AUTHOR]

Boron, as borate (or boric acid), is known as a mediator of the synthesis of ribose, ribonucleosides, and ribonucleotides (precursors of RNA) under plausible prebiotic conditions. With regard to these phenomena, the potential participation of this chemical element (as a constituent of minerals or hydrogels) for the emergence of prebiological homochirality is considered. This hypothesis is based on characteristics of crystalline surfaces as well as solubility of some minerals of boron in water or specific features of hydrogels with ester bonds from reaction of ribonucleosides and borate. [ABSTRACT FROM AUTHOR]

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5' to 3' phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive-active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.

The central dogma of molecular biology rests on two kinds of asymmetry between genomes and enzymes: informatic asymmetry, where information flows from genomes to enzymes but not from enzymes to genomes; and catalytic asymmetry, where enzymes provide chemical catalysis but genomes do not. How did these asymmetries originate? Here, we show that these asymmetries can spontaneously arise from conflict between selection at the molecular level and selection at the cellular level. We developed a model consisting of a population of protocells, each containing a population of replicating catalytic molecules. The molecules are assumed to face a trade-off between serving as catalysts and serving as templates. This trade-off causes conflicting multilevel selection: serving as catalysts is favoured by selection between protocells, whereas serving as templates is favoured by selection between molecules within protocells. This conflict induces informatic and catalytic symmetry breaking, whereby the molecules differentiate into genomes and enzymes, establishing the central dogma. We show mathematically that the symmetry breaking is caused by a positive feedback between Fisher's reproductive values and the relative impact of selection at different levels. This feedback induces a division of labour between genomes and enzymes, provided variation at the molecular level is sufficiently large relative to variation at the cellular level, a condition that is expected to hinder the evolution of altruism. Taken together, our results suggest that the central dogma is a logical consequence of conflicting multilevel selection. [ABSTRACT FROM AUTHOR]

Abstract Ribose-borate esters or Borate-Bound Species (BBS) with ribose were studied in aqueous solution by NMR and infrared (ATR) spectroscopies, as well as by DSC, PXRD and infrared spectroscopy (DRIFT) after mixing d -ribose and borate by mechanochemical procedures. NMR titrations of solutions of d -ribose by borate and borate by d -ribose were carried out, followed by 1H and 11B NMR analysis, revealing that an excess of d -ribose (relative to borate) leads to the formation of diesters and monoesters; both types of esters are stable under the studied conditions. The occurrence of furanose and the number of chemical bonds to the boron atom were confirmed by infrared spectroscopy. Interaction of Na+ with BBS revealing an ionic character was clarified by 23Na NMR spectroscopy. The samples obtained by mechanochemical procedures were amorphous, and with excess of borate present, increase their thermal stability at least up to 140 °C. These results expand the conditions of BBS as reagents in prebiotic reactions. Additionally, BBS exhibit uncommon chemical characteristics. Graphical abstract Image 1 Highlights • Ribose-borate esters as prebiological reagents. • Dry ribose-borate esters with ribose have thermal stability at least up to 140 °C. • Ribose-borate esters are amorphous and have ionic interaction with Na+. [ABSTRACT FROM AUTHOR]

The article focuses on the Origins of Life Workshop and the evidence that suggests an impact event spawned life on Earth. Topics include Origins of Life Workshop organizer, Steven Benner, a researcher at the Foundation for Applied Molecular Evolution in Alachua, Florida, and the theories presented on what gave rise to RNA (ribose nucleic acid).

Highlights • META RNA profiling is a targeted RNA sequencing method optimized for high throughput. • Both coding and non-coding RNAs can be quantified, including short microRNAs. • Early sample pooling is possible by adding sample-specific tags during RT. • Early sample pooling simplifies workflow and reduces experimental variability. • Low read depth is required since end-point PCR produces even read counts among RNAs. Abstract META RNA profiling is a simple and inexpensive method to measure the expression of multiple targeted RNAs across many samples. By assigning sample-specific tags up-front during reverse-transcription, cDNAs from multiple samples can be pooled prior to amplification and deep sequencing. Such early parallelization of samples simplifies the workflow, minimizes cross-sample experimental variability, and reduces reagent and sequencing costs. Herein we describe the theoretical framework of the method and provide a detailed protocol to facilitate its implementation. [ABSTRACT FROM AUTHOR]

Highlights • Different methodologies for the analysis and integration of diverse types of data. • Detailed studies of both RNA-Seq and miRNA-Seq data. • Identification of miRNA interactions that regulate gene expression. • Functional analyses of expression data based on a statistical correlation analysis. Abstract Messenger RNAs (mRNAs) fulfil specific biological roles in cells and, thus, their expression may be adapted to suit specific circumstances. This is in part achieved through selective gene transcription and post-transcriptional events, the regulation of which must be tightly integrated and controlled. To comprehensively study the coordinated effects of transcriptional and post-transcriptional regulatory elements, and to obtain coherent results, it is advisable to use different methodologies. Adequately integrating the data derived from these distinct methodologies then becomes critical to elucidating the relationships between the coordinated cellular effects assayed, particularly when applied to normal and disease states. Such integrated studies are likely to be particularly useful to identify markers suitable for early detection of diseases and to devise strategies for therapeutic interventions. Throughout this chapter, we will focus on the methods currently available to analyse mRNA and microRNA (miRNA) expression, paying special attention to the influence of miRNAs on mRNA metabolism. We will introduce miARma-Seq, a comprehensive pipeline that facilitates the simultaneous integration of mRNA and miRNA expression data. For illustrative purposes, we include a case study that incorporates data from RNASeq and small-RNASeq, detailing all the steps necessary to define the differential expression of both mRNA- and miRNA-encoding genes. Finally, we explore the possible regulatory relationships that drive significant and potentially relevant changes in mRNA and miRNA gene expression. [ABSTRACT FROM AUTHOR]

RNA G-quadruplexes (rG4) are stable non-canonical secondary structures composed of G-rich sequences. Many rG4 structures located in the 5’UTRs of mRNAs act as translation repressors due to their high stability which is thought to impede ribosomal scanning. That said, it is not known if these are mRNA-specific examples, or if they are indicative of a global expression regulation mechanism of the mRNAs involved in a common pathway based on structure folding recognition. Gene-ontology analysis of mRNAs bearing a predicted rG4 motif in their 5’UTRs revealed an enrichment for mRNAs associated with the colorectal cancer pathway. Bioinformatic tools for rG4 prediction, and experimental in vitro validations were used to confirm and compare the folding of the predicted rG4s of the mRNAs associated with dysregulated pathways in colorectal cancer. The rG4 folding was confirmed for the first time for 9 mRNAs. A repressive effect of 3 rG4 candidates on the expression of a reporter gene was also measured in colorectal cancer cell lines. This work highlights the fact that rG4 prediction is not yet accurate, and that experimental characterization is still essential in order to identify the precise rG4 folding sequences and the possible common features shared between the rG4 overrepresented in important biological pathways. [ABSTRACT FROM AUTHOR]

Background: To explore long non-coding RNA (lncRNA), mRNA and circular RNA (circRNA) expression profiles and their biological functions in the pathogenesis of kidney stones in ethylene glycol-induced urolithiasis rats. Results: The expression of 1440 lncRNAs, 2455 mRNAs and 145 circRNAs was altered in the kidneys of urolithiasis rats. GO and KEGG biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs, circRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on correlation analysis between differentially expressed RNAs. mRNAs coexpressed with lncRNAs were involved in many kidney diseases, e.g., Ephb6 was associated with the reabsorption ability of the kidney. Arl5b was associated with the dynamic changes in the podocyte foot process in podocyte injury. miRNAs co-expressed with circRNAs, such as rno-miR-138-5p and rno-miR-672-5p, have been proven to be functional in hypercalciuria urolithiasis. Conclusion: The expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of kidney stones. Differentially expressed lncRNAs and circRNAs might serve as treatment targets for kidney stones. [ABSTRACT FROM AUTHOR]

The origin of translation is critical for understanding the evolution of life, including the origins of life. The canonical genetic code is one of the most dominant aspects of life on this planet, while the origin of heredity is one of the key evolutionary transitions in living world. Why the translation apparatus evolved is one of the enduring mysteries of molecular biology. Assuming the hypothesis, that during the emergence of life evolution had to first involve autocatalytic systems which only subsequently acquired the capacity of genetic heredity, we propose and discuss possible mechanisms, basic aspects of the emergence and subsequent molecular evolution of translation and ribosomes, as well as enzymes as we know them today. It is possible, in this sense, to view the ribosome as a digital-to-analogue information converter. The proposed mechanism is based on the abilities and tendencies of short RNA and polypeptides to fold and to catalyse biochemical reactions. The proposed mechanism is in concordance with the hypothesis of a possible chemical co-evolution of RNA and proteins in the origin of the genetic code or even more generally at the early evolution of life on Earth. The possible abundance and availability of monomers at prebiotic conditions are considered in the mechanism. The hypothesis that early polypeptides were folding on the RNA scaffold is also considered and mutualism in molecular evolutionary development of RNA and peptides is favoured. [ABSTRACT FROM AUTHOR]

R3C ligase ribozyme catalyzes the nucleophilic attack by a 3'-hydroxyl on a 5'-α-phosphorus of triphosphates to form a 3'-5'-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a "switch" to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world. [ABSTRACT FROM AUTHOR]

Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment) approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we live in a New RNA World.

Human LSm14A is a key component of processing body (P-body) assembly that mediates interferon-β (IFN-β) production by sensing viral RNA or DNA. To the best of our knowledge, we are the first to report duck LSm14A (duLSm14A) cloning from duck embryo fibroblasts (DEFs). Full-length duLSm14A encoded 461 amino acids and was highly homologous with chicken and swan goose sequences. More interestingly, the duLSm14A mRNA was extensively expressed in all the studied tissues. In DEFs, duLSm14A was localized in the cytoplasm as P-body-like dots. Expression of duLSm14A induced IFN-β through the activation of interferon regulatory factor-1 and nuclear factor-κB in DEFs. Furthermore, knockdown of duLSm14A by small interfering RNA notably decreased poly(I:C)- or duck reovirus-induced IFN-β production. The present study results indicate that the duLSm14A is an essential sensor that mediates duck innate immunity against viral infections. [ABSTRACT FROM AUTHOR]

Ostreid herpes virus causes serious disease in the Pacific oyster ( Crassostrea gigas ), but not in the Sydney Rock Oyster ( Saccostrea glomerata ). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas , and that the gill tissue is an important site of dsRNA processing and response. [ABSTRACT FROM AUTHOR]

The viral accommodation hypothesis proposes that endogenous viral elements (EVE) from both RNA and DNA viruses are being continually integrated into the shrimp genome by natural host processes and that they can result in tolerance to viral infection by fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we hypothesized that previously reported microarray results for the presence of white spot syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427 in a domesticated giant tiger shrimp ( Penaeus monodon ) breeding stock might have represented expression from EVE, since the stock had shown uninterrupted freedom from white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens from a current stock generation were confirmed for freedom from WSSV infection using two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128 specimens (26%) positive for at least one of the ORF at very high sequence identity (95–99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated RNA templates. This revealed variable RNA expression in individual shrimp including no RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2) or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens producing putative imRNA. The variable types and numbers of the EVE and the variable RNA expression (including potential imRNA) support predictions of the viral accommodation hypothesis that EVE are randomly produced and expressed. Positive nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp sperm (germ cells), indicated that they were heritable. [ABSTRACT FROM AUTHOR]

The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas , resulting in the identification of 792 gene models containing complement-related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C-type lectins and 42 fibrinogen-related proteins. mRNAs encoding 18 serine protease domain-containing (SPC) proteins, 4 MACPF-domain containing proteins and 11 C3 receptor-like proteins were up-regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3-like receptors with integrin-α/β domain mediated the phagocytosis of C3-labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi-component complement system in C. gigas . [ABSTRACT FROM AUTHOR]

Knowledge of the chromatin transcription intermediate structures can shed light on the functioning of RNA polymerase in the cell, which is important both for basic science and for the development of therapeutic approaches for the treatment of diseases associated with transcription disorders. In this study, we used affinity monolayers formed by lipids bound to Ni to concentrate RNA polymerase from a dilute solution containing glycerol. It has been shown that RNA polymerase can be isolated and concentrated on a Ni-lipid monolayer from solutions containing cryoprotectants, which usually make it difficult to visualize samples by electron microscopy. This method of isolation allowed us to obtain more diverse spatial orientations of molecules on carbon film, which facilitates three-dimensional reconstruction from TEM data. The results obtained can be used in further structural studies of the transcription processes to identify interactions in transcriptional complexes containing RNA polymerase. [ABSTRACT FROM AUTHOR]

A novel, 10 kb RNA virus--tentatively named 'Abisko virus'--was discovered in the transcriptome data of a diseased autumnal moth (Epirrita autumnata) larva, as part of a search for the possible causes of the cyclical nature and mortality associated with geometrid moth dynamics and outbreaks in northern Fennoscandia. Abisko virus has a genome organization similar to that of the insect-infecting negeviruses, but phylogenetic and compositional bias analyses also reveal strong affiliations with plant-infecting viruses, such that both the primary host origin and taxonomic identity of the virus remain in doubt. In an extensive set of larval, pupal, and adult autumnal moth and winter moth (Operophtera brumata) outbreak samples, the virus was only detected in a few adult E. autumnata moths as well as the single larval transcriptome. The Abisko virus is therefore unlikely to be a factor in the Fennoscandia geometrid population dynamics. [ABSTRACT FROM AUTHOR]

Although viral RNA or infectious virions have been detected in the feces of individuals infected with human influenza A and B viruses (IAV/IBV), the mechanism of viral survival in the gastrointestinal tract remains unclear. We developed a model that attempts to recapitulate the conditions encountered by a swallowed virus. While IAV/IBV are vulnerable to simulated digestive juices (gastric acid and bile/pancreatic juice), highly viscous mucus protects viral RNA and virions, allowing the virus to retain its infectivity. Our results suggest that virions and RNA present in swallowed mucus are not inactivated or degraded by the gastrointestinal environment, allowing their detection in feces. [ABSTRACT FROM AUTHOR]

Extracellular vesicles are particles in mammalian body fluids that have attracted considerable attention as biomarkers for various diseases. In the present study, the authors isolated RNA aptamers with an affinity for extracellular vesicles from two library pools that encoded randomized sequences of different lengths. After the several rounds of selection, two conserved motifs are identified in the sequences that are obtained by next-generation sequencing. Most of the sequences were predicted to adopt a secondary structure that consisted of a non-conserved stem structure and a conserved loop sequence. Two minimal similar sequences are synthesized and confirmed the ability of these sequences to bind to extracellular vesicles. Circular dichroism spectroscopy and melting temperature analysis demonstrated that the aptamers were able to form a G-quadruplex structure in their loop regions and these structures were stabilized by potassium ions. Consistent with these structural data, the affinity of each aptamer for extracellular vesicles was dependent on potassium ions. The aptamers that were identified may be useful molecular tools for the development of diagnostic methods that utilize body fluids, such as blood, saliva and urine. [ABSTRACT FROM AUTHOR]

Background: Recent genome-wide profiling by sequencing and distinctive chromatin signatures has identified thousands of long non-coding RNA (lncRNA) species (>200 nt). LncRNAs have emerged as important regulators of gene expression, involving in both developmental and pathological processes. While altered expression of lncRNAs has been observed in breast cancer development, their roles in breast cancer progression and metastasis are still poorly understood.Methods: To identify novel breast cancer-associated lncRNA candidates, we employed a high-density SNP array-based approach to uncover intergenic lncRNA genes that are aberrantly expressed in breast cancer. We first evaluated the potential value as a breast cancer prognostic biomarker for one breast cancer-associated lncRNA, LincIN, using a breast cancer cohort retrieved from The Cancer Genome Atlas (TCGA) Data Portal. Then we characterized the role of LincIN in breast cancer progression and metastasis by in vitro invasion assay and a mouse tail vein injection metastasis model. To study the action of LincIN, we identified LincIN-interacting protein partner(s) by RNA pull-down experiments followed with protein identification by mass spectrometry.Results: High levels of LincIN expression are frequently observed in tumors compared to adjacent normal tissues, and are strongly associated with aggressive breast cancer. Importantly, analysis of TCGA data further suggest that high expression of LincIN is associated with poor overall survival in patients with breast cancer (P = 0.044 and P = 0.011 after adjustment for age). The functional experiments demonstrate that knockdown of LincIN inhibits tumor cell migration and invasion in vitro, which is supported by the results of transcriptome analysis in the LincIN-knockdown cells. Furthermore, knockdown of LincIN diminishes lung metastasis in a mouse tail vein injection model. We also identified a LincIN-binding protein, NF90, through which overexpression of LincIN may repress p21 protein expression by inhibiting its translation, and upregulation of p21 by LincIN knockdown may be associated with less aggressive metastasis phenotypes.Conclusions: Our studies provide clear evidence to support LincIN as a new regulator of tumor progression-metastasis at both transcriptional and translational levels and as a promising prognostic biomarker for breast cancer. [ABSTRACT FROM AUTHOR]

Background: RNA-Seq is now widely used as a research tool. Choices must be made whether to use paired-end (PE) or single-end (SE) sequencing, and whether to use strand-specific or non-specific (NS) library preparation kits. To date there has been no analysis of the effect of these choices on identifying differentially expressed genes (DEGs) between controls and treated samples and on downstream functional analysis. Results: We undertook four mammalian transcriptomics experiments to compare the effect of SE and PE protocols on read mapping, feature counting, identification of DEGs and functional analysis. For three of these experiments we also compared a non-stranded (NS) and a strand-specific approach to mapping the paired-end data. SE mapping resulted in a reduced number of reads mapped to features, in all four experiments, and lower read count per gene. Up to 4.3% of genes in the SE data and up to 12.3% of genes in the NS data had read counts which were significantly different compared to the PE data. Comparison of DEGs showed the presence of false positives (average 5%, using voom) and false negatives (average 5%, using voom) using the SE reads. These increased further, by one or two percentage points, with the NS data. Gene ontology functional enrichment (GO) of the DEGs arising from SE or NS approaches, revealed striking differences in the top 20 GO terms, with as little as 40% concordance with PE results. Caution is therefore advised in the interpretation of such results. By comparison, there was overall consistency in gene set enrichment analysis results. Conclusions: A strand-specific protocol should be used in library preparation to generate the most reliable and accurate profile of expression. Ideally PE reads are also recommended particularly for transcriptome assembly. Whilst SE reads produce a DEG list with around 5% of false positives and false negatives, this method can substantially reduce sequencing cost and this saving could be used to increase the number of biological replicates thereby increasing the power of the experiment. As SE reads, when used in association with gene set enrichment, can generate accurate biological results, this may be a desirable trade-off. [ABSTRACT FROM AUTHOR]

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation. [ABSTRACT FROM AUTHOR]

In eukaryotic cells tRNA synthesis is negatively regulated by the protein Maf1, conserved from yeast to humans. Maf1 from yeast Saccharomyces cerevisiae mediates repression of trna transcription when cells are transferred from medium with glucose to medium with glycerol, a non-fermentable carbon source. The strain with deleted gene encoding Maf1 ( maf1 Δ) is viable but accumulates tRNA precursors. In this study tRNA precursors were analysed by RNA-Seq and Northern hybridization in wild type strain and maf1 Δ mutant grown in glucose medium or upon shift to repressive conditions. A negative effect of maf1 Δ mutant on the addition of the auxiliary CCA nucleotides to the 3′ end of pre-tRNAs was observed in cells shifted to unfavourable growth conditions. This effect was reduced by overexpression of the yeast CCA1 gene encoding ATP(CTP):tRNA nucleotidyltransferase. The CCA sequence at the 3′ end is important for export of tRNA precursors from the nucleus and essential for tRNA charging with amino acids. Data presented here indicate that CCA-addition to intron-containing end-processed tRNA precursors is a limiting step in tRNA maturation when there is no Maf1 mediated RNA polymerase III (Pol III) repression. The correlation between CCA synthesis and Pol III regulation by Maf1 could be important in coordination of tRNA transcription, processing and regulation of translation. [ABSTRACT FROM AUTHOR]

Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III. [ABSTRACT FROM AUTHOR]

In this study, we reported two featured series of rRNA-derived RNA fragments (rRFs) from the small RNA sequencing (sRNA-seq) data of Amblyomma testudinarium using the Illunima platform. Two series of rRFs (rRF5 and rRF3) were precisely aligned to the 5' and 3' ends of the 5.8S and 28S rRNA gene. The rRF5 and rRF3 series were significantly more highly expressed than the rRFs located in the body of the rRNA genes. These series contained perfectly aligned reads, the lengths of which varied progressively with 1-bp differences. The rRF5 and rRF3 series in the same expression pattern exist ubiquitously from ticks to human. The cellular experiments showed the RNAi knockdown of one 20-nt rRF3 induced the cell apoptosis and inhibited the cell proliferation. In addition, the RNAi knockdown resulted in a significant decrease of H1299 cells in the G2 phase of the cell cycle. These results indicated the rRF5 and rRF3 series were not random intermediates or products during rRNA degradation, but could constitute a new class of small RNAs that deserves further investigation. [ABSTRACT FROM AUTHOR]

The dominant paradigm in origin of life research is that of an RNA world. However, despite experimental progress towards the spontaneous formation of RNA, the RNA world hypothesis still has its problems. Here, we introduce a novel computational model of chemical reaction networks based on RNA secondary structure and analyze the existence of autocatalytic sub-networks in random instances of this model, by combining two well-established computational tools. Our main results are that (i) autocatalytic sets are highly likely to exist, even for very small reaction networks and short RNA sequences, and (ii) sequence diversity seems to be a more important factor in the formation of autocatalytic sets than sequence length. These findings could shed new light on the probability of the spontaneous emergence of an RNA world as a network of mutually collaborative ribozymes. [ABSTRACT FROM AUTHOR]

The basic steps of protein synthesis are carried out by the ribosome, a very large and complex ribonucleoprotein particle. In keeping with its proposed emergence from an RNA world, all three of its core mechanisms--amino-acyl-tRNA selection, catalysis of peptide bond formation and coupled translocation of mRNA and tRNA--are embodied in the properties of ribosomal RNA, while its proteins play a supportive role. This article is part of the themed issue 'Perspectives on the ribosome'. [ABSTRACT FROM AUTHOR]

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo. [ABSTRACT FROM AUTHOR]