Almost all biomedical research to date has relied upon mean measurements from cell populations, however it is well established that what it is observed at this macroscopic level can be the result of many interactions of several different single cells. Thus, the observable macroscopic ‘average’ cannot outright be used as representative of the ‘average cell’. Rather, it is the resulting emerging behaviour of the actions and interactions of many different cells. Single‐cell RNA sequencing (scRNA‐Seq) enables the comparison of the (...) transcriptomes of individual cells. This provides high‐resolution maps of the dynamic cellular programmes allowing us to answer fundamental biological questions on their function and evolution. It also allows to address medical questions such as the role of rare cell populations contributing to disease progression and therapeutic resistance. Furthermore, it provides an understanding of context‐specific dependencies, namely the behaviour and function that a cell has in a specific context, which can be crucial to understand some complex diseases, such as diabetes, cardiovascular disease and cancer. Here, we provide an overview of scRNA‐Seq, including a comparative review of emerging technologies and computational pipelines. We discuss the current and emerging applications and focus on tumour heterogeneity a clear example of how scRNA‐Seq can provide new understanding of a complex disease. Additionally, we review the limitations and highlight the need of powerful computational pipelines and reproducible protocols for the broader acceptance of this technique in basic and clinical research. (shrink)

Mapping the paths that stem and progenitor cells take en route to differentiate and elucidating the underlying molecular controls are key goals in developmental and stem cell biology. However, with population level analyses it is difficult − if not impossible − to define the transition states and lineage trajectory branch points within complex developmental lineages. Single‐cell RNA‐sequencing analysis can discriminate heterogeneity in a population of cells and even identify rare or transient intermediates. In this review, we propose that using (...) these data, one can infer the lineage trajectories of individual stem cells and identify putative branch points. Clonal lineage tracing of stem cells allows one to define the outcome of differentiation. Integrating these single cell‐based approaches provides a robust strategy for establishing and testing models of how an individual stem cell changes through time to differentiate and self‐renew. (shrink)

Eukaryotic mRNAs are monocistronic, and therefore mechanisms exist that coordinate the synthesis of multiprotein complexes in order to obtain proper stoichiometry at the appropriate intracellular locations. RNA‐binding proteins containing low‐complexity sequences are prone to generate liquid droplets via liquid‐liquid phase separation, and in this way create cytoplasmic assemblages of functionally related mRNAs. In a recent iCLIP study, we showed that the Drosophila RNA‐binding protein Imp, which exhibits a C‐terminal low‐complexity sequence, increases the formation of F‐actin by binding to 3′ untranslated (...) regions of mRNAs encoding components participating in F‐actin biogenesis. We hypothesize that phase transition is a mechanism the cell employs to increase the local mRNA concentration considerably, and in this way synchronize protein production in cytoplasmic territories, as discussed in the present review. (shrink)

The folding of RNA sequences into secondary structures is a simple yet biophysically grounded model of a genotype–phenotype map. Its computational and mathematical analysis has uncovered a surprisingly rich statistical structure characterized by shape space covering, neutral networks and plastogenetic congruence. I review these concepts and discuss their evolutionary implications. BioEssays 24:1164–1177, 2002. © 2002 Wiley‐Periodicals, Inc.

Fusion genes formed by chromosomal rearrangements are common drivers of cancer. Recent innovations in the field of next‐generation sequencing (NGS) have seen a dynamic shift from traditional fusion detection approaches, such as visual characterization by fluorescence, to more precise multiplexed methods. There are many different NGS‐based approaches to fusion gene detection and deciding on the most appropriate method can be difficult. Beyond the experimental approach, consideration needs to be given to factors such as the ease of implementation, processing time, (...) associated costs, and the level of expertise required for data analysis. Here, the different NGS‐based methods for fusion gene detection, the basic principles underlying the techniques, and the benefits and limitations of each approach are reviewed. This article concludes with a discussion of how NGS will impact fusion gene detection in a clinical context and from where the next innovations are evolving. (shrink)

RNA polymerase II is recruited to DNA double‐strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage‐induced long non‐coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA‐like molecules or degraded by different ribonucleases. They can also form double‐stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to (...) the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR. (shrink)

The aim of the study is to analyze viruses using parameters obtained from distributions of nucleotide sequences in the viral RNA. Seeking for the input data homogeneity, we analyze single-stranded RNA viruses only. Two approaches are used to obtain the nucleotide sequences; In the first one, chunks of equal length are considered. In the second approach, the whole RNA genome is divided into parts by adenine or the most frequent nucleotide as a “space”. Rank–frequency distributions are studied in both cases. (...) The defined nucleotide sequences are signs comparable to a certain extent to syllables or words as seen from the nature of their rank–frequency distributions. Within the first approach, the Pólya and the negative hypergeometric distribution yield the best fit. For the distributions obtained within the second approach, we have calculated a set of parameters, including entropy, mean sequence length, and its dispersion. The calculated parameters became the basis for the classification of viruses. We observed that proximity of viruses on planes spanned on various pairs of parameters corresponds to related species. In certain cases, such a proximity is observed for unrelated species as well calling thus for the expansion of the set of parameters used in the classification. We also observed that the fifth most frequent nucleotide sequences obtained within the second approach are of different nature in case of human coronaviruses. We expect that our findings will be useful as a supplementary tool in the classification of diseases caused by RNA viruses with respect to severity and contagiousness. (shrink)

RNA editing is a newly described genetic phenomenon. It encompasses widely different molecular mechanisms and events. According to the specific RNA modification, RNA editing can be broadly classified into six major types. Type II RNA editing occurs in plants and mammals; it consists predominantly in cytidine to uridine conversions resulting from deamination/transamination or transglycosylation, although in plants other mechanisms have not been excluded. Apolipoprotein B mRNA editing is the only well‐documented editing phenomenon in mammals. It is an intranuclear event that (...) occurs posttranscriptionally, coincident with splicing and polyadenylation. Recent observations indicate that the tissue‐ and sequence‐specific process is mediated by an enzyme that has separate domains for editing and sequence recognition. The presence of apolipoprotein B mRNA editing activity in tissues that do not produce the protein suggests that other RNAs may be edited and RNA editing may be a genetic phenomenon of general biological importance to the cell. (shrink)

The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück's phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA (mRNA), genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA (...) due to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers’ laboratory at Ghent University (Belgium) and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his ‘RNA phage lab’ with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s. (shrink)

The advent of deep sequencing technology has unexpectedly advanced our structural understanding of molecules composed of nucleic acids. A significant amount of progress has been made recently extrapolating the chemical methods to probe RNA structure into sequencing methods. Herein we review some of the canonical methods to analyze RNA structure, and then we outline how these have been used to probe the structure of many RNAs in parallel. The key is the transformation of structural biology problems into (...) class='Hi'>sequencing problems, whereby sequencing power can be interpreted to understand nucleic acid proximity, nucleic acid conformation, or nucleic acid‐protein interactions. Utilizing such technologies in this way has the promise to provide novel structural insights into the mechanisms that control normal cellular physiology and provide insight into how structure could be perturbed in disease. (shrink)

Genetic variability is considered a key to the evolvability of species. The conversion of an adenosine (A) to inosine (I) in primary RNA transcripts can result in an amino acid change in the encoded protein, a change in secondary structure of the RNA, creation or destruction of a splice consensus site, or otherwise alter RNA fate. Substantial transcriptome and proteome variability is generated by A‐to‐I RNA editing through site‐selective post‐transcriptional recoding of single nucleotides. We posit that this epigenetic source of (...) phenotypic variation is an unrecognized mechanism of adaptive evolution. The genetic variation introduced through editing occurs at low evolutionary cost since predominant production of the wild‐type protein is retained. This property even allows exploration of sequence space that is inaccessible through mutation, leading to increased phenotypic plasticity and provides an evolutionary advantage for acclimatization as well as long‐term adaptation. Furthermore, continuous probing for novel RNA editing sites throughout the transcriptome is an intrinsic property of the editing machinery and represents the molecular basis for increased adaptability. We propose that higher organisms have therefore evolved to systems with increasing RNA editing activity and, as a result, to more complex systems. (shrink)

Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre‐exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF65 fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA (...) binding to U2AF65 may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF65 might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3′ splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage. (shrink)

In eukaryotic cells intron sequences are usually spliced out with a high degree of precision from heterogenous nuclear RNA (hnRNA) to give functional mRNA with exons in their right order. Provided with the right substrates, cell extracts can achieve the same. With exotic substrates, on the other hand, the same extracts can cut exons from one RNA and join them to exons from another RNA, a process termed trans‐splicing. In vivo, RNA trans‐splicing could lead to faulty, but also to novel (...) proteins and, through reverse transcription, to genomic rearrangements. So far, in living cells trans‐splicing has only been invoked to occur in trypanosomes. In these unicellular organisms most if not all mRNAs are made discontinuously, so that the mature mRNAs carry, at their 5′ end, a common 35‐nucleotide sequence, called a miniexon, encoded separately in the genome. The miniexon is most likely joined to the body of the mRNA by trans‐splicing. (shrink)

hiCLIP (RNA hybrid and individual‐nucleotide resolution ultraviolet cross‐linking and immunoprecipitation), is a novel technique developed by Sugimoto et al. (2015). Here, the use of different adaptors permits a controlled ligation of the two strands of a RNA duplex allowing the identification of each arm in the duplex upon sequencing. The authors chose a notoriously difficult to study double‐stranded RNA‐binding protein (dsRBP) termed Staufen1, a mammalian homolog of Drosophila Staufen involved in mRNA localization and translational control. Using hiCLIP, they discovered (...) a dominance of intramolecular RNA duplexes compared to the total RNA duplexes identified. Importantly, the authors discovered two different types of intramolecular duplexes in the cell: highly translated mRNAs with long‐range duplexes in their 3′‐UTRs and poorly translated mRNAs with duplexes in their coding region. In conclusion, the authors establish hiCLIP as an important novel technique for the identification of RNA secondary structures that serve as in vivo binding sites for dsRBPs. (shrink)

RNA binding proteins (RBPs) are key factors for the regulation of gene expression by binding to cis elements, i.e. short sequence motifs in RNAs. Recent studies demonstrate that cooperative binding of multiple RBPs is important for the sequence‐specific recognition of RNA and thereby enables the regulation of diverse biological activities by a limited set of RBPs. Cross‐linking immuno‐precipitation (CLIP) and other recently developed high‐throughput methods provide comprehensive, genome‐wide maps of protein‐RNA interactions in the cell. Structural biology gives detailed insights into (...) molecular mechanisms and principles of RNA recognition by RBPs, but has so far focused on single RNA binding proteins and often on single RNA binding domains. The combination of high‐throughput methods and detailed structural biology studies is expected to greatly advance our understanding of the code for protein‐RNA recognition in gene regulation, as we review in this article. (shrink)

In recent years, sequencing technologies have enabled the identification of a wide range of non-coding RNAs (ncRNAs). Unfortunately, annotation and integration of ncRNA data has lagged behind their identification. Given the large quantity of information being obtained in this area, there emerges an urgent need to integrate what is being discovered by a broad range of relevant communities. To this end, the Non-Coding RNA Ontology (NCRO) is being developed to provide a systematically structured and precisely defined controlled vocabulary for (...) the domain of ncRNAs, thereby facilitating the discovery, curation, analysis, exchange, and reasoning of data about structures of ncRNAs, their molecular and cellular functions, and their impacts upon phenotypes. The goal of NCRO is to serve as a common resource for annotations of diverse research in a way that will significantly enhance integrative and comparative analysis of the myriad resources currently housed in disparate sources. It is our belief that the NCRO ontology can perform an important role in the comprehensive unification of ncRNA biology and, indeed, fill a critical gap in both the Open Biological and Biomedical Ontologies (OBO) Library and the National Center for Biomedical Ontology (NCBO) BioPortal. Our initial focus is on the ontological representation of small regulatory ncRNAs, which we see as the first step in providing a resource for the annotation of data about all forms of ncRNAs. The NCRO ontology is free and open to all users. (shrink)

In organisms, ribonucleic acid plays an essential role. Its function is being discovered more and more. Due to the conserved nature of RNA sequences, its function mainly depends on the RNA secondary structure. The discovery of an approximate relationship between two RNA secondary structures helps to understand their functional relationship better. It is an important and urgent task to explore structural similarities from the graphical representation of RNA secondary structures. In this paper, a novel graphical analysis method based on the (...) triple vector curve representation of RNA secondary structures is proposed. A combinational method involving a discrete wavelet transform and fractal dimension with sliding window is introduced to analyze and compare the graphs derived from feature extraction; after that, the distance matrix is generated. Then, the distance matrix is analyzed by clustering and visualized as a clustering tree. RNA virus and noncoding RNA datasets are applied to perform experiments and analyze the clustering tree. The results show that the proposed method yields more accurate results in the comparison of RNA secondary structures. (shrink)

The only RNA molecules known to be branched are circular structures with tails known as lariats that arise during nuclear pre‐mRNA splicing. Lariats accumulate within a large multicomponent particle called a spliceosome that forms upon the addition of unspliced mRNA to nuclear extracts. Recently an RNA molecule has been observed to catalyze branch formation. In this case a single intron of a yeast mitochondrial pre‐mRNA participates in a self‐splicing reaction that results in the accumulation of branched lariats that are processed (...) to correctly spliced exons.An enzyme highly specific for branch removal found in the same extracts that form branches during pre‐mRNA splicing can debranch RNA lariats to their linear forms without loss of nucleotides.The chemical synthesis of branched RNA has recently been achieved. High yields of sequence‐specific oligonucleotides are now available for the analysis of RNA splicing by techniques dependent on branch‐site recognition. (shrink)

The role of RNA as a messenger in the expression of the genome has been long appreciated, but its functions in regulating chromatin and chromosome structure are no less interesting. Recent results have shown that small RNAs guide chromatin‐modifying complexes to chromosomal regions in a sequence‐specific manner to elicit transcriptional repression. However, sequence‐specific targeting by means of base pairing seems to be only one mechanism by which RNA is employed for epigenetic regulation. The focus of this review is on large (...) RNAs that act in the dosage‐compensation pathways of flies and mammals. These RNAs associate with chromatin over the length of whole chromosomes and are crucial for spreading epigenetic changes in chromatin structure. They do not appear to act in a sequence‐specific manner but might provide scaffolds for co‐operative binding of chromatin‐associated complexes that enable spreading of chromatin modifications. BioEssays 25:434–442, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

New DNA sequencing technologies have provided novel insights into eukaryotic genomes, epigenomes, and the transcriptome, including the identification of new non‐coding RNA (ncRNA) classes such as promoter‐associated RNAs and long RNAs. Moreover, it is now clear that up to 90% of eukaryotic genomes are transcribed, generating an extraordinary range of RNAs with no coding capacity. Taken together, these new discoveries are modifying the status quo in genomic science by demonstrating that the eukaryotic gene pool is divided into two distinct (...) categories of transcripts: protein‐coding and non‐coding. The function of the majority of ncRNAs produced by the transcriptome is largely unknown; however, it is probable that many are associated with epigenetic mechanisms. The purpose of this review is to describe the most recent discoveries in the ncRNA field that implicate these molecules as key players in the epigenome. (shrink)

The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück’s phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA, genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA due (...) to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers’ laboratory at Ghent University and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his ‘RNA phage lab’ with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s. (shrink)

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill (...) knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed. (shrink)

Cells contain a myriad of membraneless ribonucleoprotein (RNP) condensates with distinct compositions of proteins and RNAs. RNP condensates participate in different cellular activities, including RNA storage, mRNA translation or decay, stress response, etc. RNP condensates are assembled via liquid‐liquid phase separation (LLPS) driven by multivalent interactions. Transition of RNP condensates into bodies with abnormal material properties, such as solid‐like amyloid structures, is associated with the pathogenesis of various diseases. In this review, we focus on how RNAs regulate multiple aspects of (...) RNP condensates, such as dynamic assembly and/or disassembly and biophysical properties. RNA properties – including concentration, sequence, length and structure – also determine the phase behaviors of RNP condensates. RNA is also involved in specifying autophagic degradation of RNP condensates. Unraveling the role of RNA in RNPs provides novel insights into pathological accumulation of RNPs in various diseases. This new understanding can potentially be harnessed to develop therapeutic strategies. (shrink)

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and doublestranded RNA into ~21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We (...) show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic. (shrink)

The human genome project's lasting legacies are the emerging insights into human physiology and disease, and the ascendance of biology as the dominant science of the 21st century. Sequencing revealed that >90% of the human genome is not coding for proteins, as originally thought, but rather is overwhelmingly transcribed into non‐protein coding, or non‐coding, RNAs (ncRNAs). This discovery initially led to the hypothesis that most genomic DNA is “junk”, a term still championed by some geneticists and evolutionary biologists. In (...) contrast, molecular biologists and biochemists studying the vast number of transcripts produced from most of this genome “junk” often surmise that these ncRNAs have biological significance. What gives? This essay contrasts the two opposing, extant viewpoints, aiming to explain their bases, which arise from distinct reference frames of the underlying scientific disciplines. Finally, it aims to reconcile these divergent mindsets in hopes of stimulating synergy between scientific fields. (shrink)

Nonsense‐mediated RNA decay (NMD) is an evolutionary conserved system of RNA surveillance that detects and degrades RNA transcripts containing nonsense mutations. Given that these mutations arise at a relatively low frequency, are there any as yet unknown substrates of NMD in a wild‐type cell? With this question in mind, Mendell et al.1 have used a microarray assay to identify those human genes under NMD regulation. Their results show that, in human cells, NMD regulates hundreds of physiologic transcripts and not just (...) those containing nonsense mutations. Among the NMD targets are a number of non‐functional RNAs expressed from vestigial sequences derived from retroviral and transposable elements. These findings support the notion that NMD is a high profile post‐transcriptional mechanism micromanaging the activity of multiple gene batteries and suppressing the expression of genetic remnants. BioEssays 27:463–466, 2005. © 2005 Wiley Periodicals, Inc. (shrink)

In eukaryotes, double‐stranded RNAs (dsRNAs) or short, interfering dsRNAs (siRNAs) can reduce the accumulation of a sequence‐related mRNA, often resulting in a loss‐of‐function phenotype—a process termed RNA interference (RNAi). Unfortunately, some mRNAs are resistant to the effects of dsRNA. Experiments designed to unravel RNAi mechanisms in Caenorhabditis elegans have led to the identification of two worm proteins, RRF‐31,2 and, now, ERI‐1,3 that can inhibit RNAi responses. Animals defective in either protein can display enhanced RNAi phenotypes for mRNAs that were previously (...) resistant to dsRNA. Since ERI‐1 is a conserved protein, development of procedures to enhance RNAi effectiveness in other systems may be possible. BioEssays 26:715–718, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

The biogenesis of RNAs and proteins is a threat to the cell. Indeed, the act of transcription and nascent RNAs challenge DNA stability. Both RNAs and nascent proteins can also initiate the formation of toxic aggregates because of their physicochemical properties. In reviewing the literature, I show that co-transcriptional and co-translational biophysical constraints can trigger DNA instability that in turn increases the likelihood that sequences that alleviate the constraints emerge over evolutionary time. These directed genetic variations rely on the biogenesis (...) of small RNAs that are transcribed directly from challenged DNA regions or processed from the transcripts that directly or indirectly generate constraints or aggregates. These small RNAs can then target the genomic regions from which they initially originate and increase the local mutation rate of the targeted loci. This mechanism is based on molecular pathways involved in anti-parasite genome defence systems, and implies that gene expression-related biophysical constraints represent a driving force of genome evolution. Are randomly generated mutations the only source of genetic variation during evolution? This paper describes the molecular mechanisms by which co-transcriptional and co-translational biophysical constraints trigger genetic variations in targeted-genomic sequences in an RNA-depending manner. This directed-mutational process that drives genome evolution is related to antiparasite genome defence systems. (shrink)

Electron micrographs of active ribosomal genes from many species show a similar picture in which gene regions covered with nascent transcripts alternate with apparently non‐transcribed spacers. Since the gradients of visible nascent transcripts stop near the 3′ end of the 28S sequence it has often been assumed that transcription by RNA polymerase I also terminates at that point. Recent biochemical studies have shown however, that transcription continues far beyond the 3′ end of the 28S and in some species continues across (...) the entire spacer. In this article we review the evidence for spacer transcription in Xenopus, mouse and Drosophila and discuss possible reasons for its invisibility in the electron microscope. (shrink)

Identification of non-coding RNAs (ncRNAs) has been significantly enhanced due to the rapid advancement in sequencing technologies. On the other hand, semantic annotation of ncRNA data lag behind their identification, and there is a great need to effectively integrate discovery from relevant communities. To this end, the Non-Coding RNA Ontology (NCRO) is being developed to provide a precisely defined ncRNA controlled vocabulary, which can fill a specific and highly needed niche in unification of ncRNA biology.

Almost all messenger RNAs carry a polyadenylate tail that is added in a post‐transcriptional reaction. In the nuclei of animal cells, the 3'‐end of the RNA is formed by endonucleolytic cleavage of the primary transcript at the site of poly (A) addition, followed by the polymerisation of the tail. The reaction depends on specific RNA sequences upstream as well as downstream of the polyadenylation site. Cleavage and polyadenylation can be uncoupled in vitro. Polyadenylation is carried out by poly(A) polymerase with (...) the aid of a specificity factor that binds the polyadenylation signal AAUAAA. Several aditional factors are required for the initial cleavage. A newly discovered poly(A)‐binding protein stimulates poly(A) tail synthesis and may be involved in the control of tail length. Polyadenylation reactions different from this scheme, either in other organisms or under special physiological circumstances, are discussed. (shrink)

Most molecular biological concepts derive from physical chemical assumptions about the genetic code that are basically more than 40 years old. Additionally, systems biology, another quantitative approach, investigates the sum of interrelations to obtain a more holistic picture of nucleotide sequence order. Recent empirical data on genetic code compositions and rearrangements by mobile genetic elements and non-coding RNAs, together with results of virus research and their role in evolution, does not really fit into these concepts and compel a re-examination. In (...) this review we try to find an alternate hypothesis. It seems plausible now that if we look at the abundance of regulatory RNAs and persistent viruses in host genomes, we will find more and more evidence that the key players that edit the genetic codes of host genomes are consortia of RNA agents and viruses that drive evolutionary novelty and regulation of cellular processes in all steps of development. This agent-based approach may lead to a qualitative RNA sociology that investigates and identifies relevant behavioural motifs of cooperative RNA consortia. In addition to molecular biological perspectives this may lead to a better understanding of genetic code evolution and dynamics. (shrink)

In plants, virus‐derived double‐stranded RNA is processed into small interfering (si)RNAs by RNAse III‐type enzymes. siRNAs are believed to guide an RNA‐induced silencing complex (RISC) to promote sequence‐specific degradation (or ‘slicing’) of homologous viral transcripts. This process, called RNA silencing, likely involves Argonaute (AGO) proteins that are known components of plant and animal RISCs. Plant viruses commonly counteract the silencing immune response by producing suppressor proteins, but the molecular basis of their action has remained largely unclear. A recent study by (...) Zhang and colleagues1 now shows that the 2b suppressor of Cucumber mosaic virus directly interacts with Arabidopsis AGO1 and inhibits its slicing activity, suggesting that AGO1 might be a component of the elusive plant antiviral RISC. BioEssays 29:319–323, 2007. © 2007 Wiley Periodicals, Inc. (shrink)

RNAs can do many things. They can store information, act in the world, and respond to the world. Because of these capabilities biologists have proposed a primordial ‘RNA world’ in which RNA, rather than DNA, performed the central role of replicator and repository of adaptive information. Deacon dismisses this hypothesis because replication is not about anything and because the structure of replicating molecules cannot contain information about the environment. I dispute both claims. An RNA and its opposite-sense complement represent each (...) other and, by two rounds of complementation, represent themselves. Although nucleic acid sequences do not change in response to their present environment, these sequences embody information about ancestral environments via the selective filtering of alternative sequences in those environments. Nucleic acid sequences are the textual record of what has worked in the past. (shrink)

The genetic code has evolved from its initial non-degenerate wobble version until reaching its present state of degeneracy. By using the stereochemical hypothesis, we revisit the problem of codon assignations to the synonymy classes of amino-acids. We obtain these classes with a simple classifier based on physico-chemical properties of nucleic bases, like hydrophobicity and molecular weight. Then we propose simple RNA ring structures that present, overlap included, one and only one codon by synonymy class as solutions of a combinatory variational (...) problem. We compare these solutions to sequences of present RNAs considered as relics, with a high interspecific invariance, like invariant parts of tRNAs and micro-RNAs. We conclude by emphasizing some optimal properties of the genetic code. (shrink)

Thomas Kuhn described the progress of science as comprising occasional paradigm shifts separated by interludes of ‘normal science’. The paradigm that has held sway since the inception of molecular biology is that genes (mainly) encode proteins. In parallel, theoreticians posited that mutation is random, inferred that most of the genome in complex organisms is non‐functional, and asserted that somatic information is not communicated to the germline. However, many anomalies appeared, particularly in plants and animals: the strange genetic phenomena of paramutation (...) and transvection; introns; repetitive sequences; a complex epigenome; lack of scaling of (protein‐coding) genes and increase in ‘noncoding’ sequences with developmental complexity; genetic loci termed ‘enhancers’ that control spatiotemporal gene expression patterns during development; and a plethora of ‘intergenic’, overlapping, antisense and intronic transcripts. These observations suggest that the original conception of genetic information was deficient and that most genes in complex organisms specify regulatory RNAs, some of which convey intergenerational information. Also see the video abstract here: https://youtu.be/qxeGwahBANw. (shrink)

Recent experimental evidence suggests that most of the genome is transcribed into non‐coding RNAs. The initial transcripts undergo further processing generating shorter, metabolically stable RNAs with diverse functions. Small nucleolar RNAs (snoRNAs) are non‐coding RNAs that modify rRNAs, tRNAs, and snRNAs that were considered stable. We review evidence that snoRNAs undergo further processing. High‐throughput sequencing and RNase protection experiments showed widespread expression of snoRNA fragments, known as snoRNA‐derived RNAs (sdRNAs). Some sdRNAs resemble miRNAs, these can associate with argonaute proteins (...) and influence translation. Other sdRNAs are longer, form complexes with hnRNPs and influence gene expression. C/D box snoRNA fragmentation patterns are conserved across multiple cell types, suggesting a processing event, rather than degradation. The loss of expression from genetic loci that generate canonical snoRNAs and processed snoRNAs results in diseases, such as Prader‐Willi Syndrome, indicating possible physiological roles for processed snoRNAs. We propose that processed snoRNAs acquire new roles in gene expression and represent a new class of regulatory RNAs distinct from canonical snoRNAs.Also watch the Video Abstract. (shrink)

Kawasaki disease (KD) is an acute self‐limiting vasculitis with coronary complications, usually occurring in children. The incidence of KD in children is increasing year by year, mainly in East Asian countries, but relatively stably in Europe and America. Although studies on KD have been reported, the pathogenesis of KD is unknown. With the development of high‐throughput sequencing technology, growing number of regulatory noncoding RNAs (ncRNAs) including microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) have been identified to (...) involved in KD. However, the role of ncRNAs in KD has not been comprehensively elucidated. Therefore, it is significative to study the regulatory role of ncRNA in KD, which might help to uncover new and effective therapeutic strategies for KD. In this review, we summarize recent studies on ncRNA in KD from the perspectives of immune disorders, inflammatory disorders, and endothelial dysfunction, and highlight the potential of ncRNAs as therapeutic targets for KD. (shrink)

Recent analysis of the contribution of replication slippage to genome evolution shows that it has played a significant role in all species from eubacteria to humans. The overall level of repetition in genomes is related to genome size and to the degree of repetition that can be measured within individual ribosomai RNA genes, suggesting that the entire genome accepts simple sequences in a concerted manner when its size increases. Although coding sequences accept simple sequences much less readily than non‐coding sequences, (...) they accept some repeats, particularly (CAG)n, preferentially. This may have consequences for the evolution of the genes involved in trinucleotide expansion diseases and the transcriptional networks of which they may form a part. (shrink)

Many viral and cellular mRNA species contain a leader sequence derived from a distant upstream site on the same gene by a process of RNA splicing. This process usually involves either nuclear functions or self‐splicing of RNA molecules. Coronavirus, a cytoplasmic RNA virus, unfolds yet another mechanism of joining RNA, which involves the use of a free leader RNA molecule. This molecule is synthesized and dissociates from the template RNA, and subsequently reassociates with the template RNA at down‐stream initiation sites (...) of subgenomic mRNAs to serve as the primer for transcription. This leader‐primed transcriptional process thus generates viral mRNAs with a fused leader sequence. A similar mechanism might also operate in the mRNA transcription of African trypanosomes. (shrink)

Theoretical minimal RNA rings attempt to mimick life’s primitive RNAs. At most 25 22-nucleotide-long RNA rings code once for each biotic amino acid, a start and a stop codon and form a stem-loop hairpin, resembling consensus tRNAs. We calculated, for each RNA ring’s 22 potential splicing positions, similarities of predicted secondary structures with tRNA vs. rRNA secondary structures. Assuming rRNAs partly derived from tRNA accretions, we predict positive associations between relative secondary structure similarities with rRNAs over tRNAs and genetic code (...) integration orders of RNA ring anticodon cognate amino acids. Analyses consider for each secondary structure all nucleotide triplets as potential anticodon. Anticodons for ancient, chemically inert cognate amino acids are most frequent in the 25 RNA rings. For RNA rings with primordial cognate amino acids according to tRNA-homology-derived anticodons, tRNA-homology and coding sequences coincide, these are separate for predicted cognate amino acids that presumably integrated late the genetic code. RNA ring secondary structure similarity with rRNA over tRNA secondary structures associates best with genetic code integration orders of anticodon cognate amino acids when assuming split anticodons (one and two nucleotides at the spliced RNA ring 5′ and 3′ extremities, respectively), and at predicted anticodon location in the spliced RNA ring’s midst. Results confirm RNA ring homologies with tRNAs and CDs, ancestral status of tRNA half genes split at anticodons, the tRNA-rRNA axis of RNA evolution, and that single theoretical minimal RNA rings potentially produce near-complete proto-tRNA sets. Hence genetic code pre-existence determines 25 short circular gene- and tRNA-like RNAs. Accounting for each potential splicing position, each RNA ring potentially translates most amino acids, realistically mimicks evolution of the tRNA-rRNA translation machinery. These RNA rings ‘of creation’ remind the uroboros’ (snake biting its tail) symbolism for creative regeneration. (shrink)

Insight into animal biology and development provided by classical genetic analysis of the model organism Drosophila melanogaster was an incentive to develop advanced genetic tools for this insect. But genetic systems for the over one million other known insect species are largely undeveloped. With increasing information about insect genomes resulting from next generation sequencing, RNA interference is now the method of choice for reverse genetics, although it is constrained by the means of delivery of interfering RNA. A recent advance (...) to ensure sustained delivery with minimal experimental intervention or trauma to the insect is to exploit commensal bacteria for symbiont‐mediated RNA interference. This technology not only offers an efficient means for RNA interference in insects in laboratory conditions, but also has potential for use in the control of human disease vectors, agricultural pests and pathogens of beneficial insects. (shrink)

Throughout evolution, there has been interaction and exchange between RNA pools in the environment, and DNA and RNA pools of eukaryotic organisms. Metagenomic and metatranscriptomic sequencing of invertebrate hosts and their microbiota has revealed a rich evolutionary history of RNA virus shuttling between species. Horizontal transfer adapted the RNA pool for successful future interactions which lead to zoonotic transmission and detrimental RNA viral pandemics like SARS‐CoV2. In eukaryotes, noncoding RNA (ncRNA) is an established mechanism derived from prokaryotes to defend (...) against viral attack through innate immunity and regulation of host‐derived mRNA. Transgenerational inheritance of ncRNA is evidence for feedforward adaptive immunity and epigenetically encoded environmental change across generations, which may explain the ‘‘missing heritability’’ of common disease. Causal graph theory and the Price Equation can model epigenetic inheritance involving dynamic internal and external RNA pools. Experimental designs should include metatranscriptomic analyses to understand how ncRNA responds to rapidly changing environmental conditions, within and between organisms, and across generations. (shrink)

The Streptococcus pyogenes CRISPR‐Cas system has gained widespread application as a genome editing and gene regulation tool as simultaneous cellular delivery of the Cas9 protein and guide RNAs enables recognition of specific DNA sequences. The recent discovery that Cas9 can also bind and cleave RNA in an RNA‐programmable manner indicates the potential utility of this system as a universal nucleic acid‐recognition technology. RNA‐targeted Cas9 (RCas9) could allow identification and manipulation of RNA substrates in live cells, empowering the study of cellular (...) gene expression, and could ultimately spawn patient‐ and disease‐specific diagnostic and therapeutic tools. Here we describe the development of RCas9 and compare it to previous methods for RNA targeting, including engineered RNA‐binding proteins and other types of CRISPR‐Cas systems. We discuss potential uses ranging from live imaging of transcriptional dynamics to patient‐specific therapies and applications in synthetic biology. (shrink)

Many innate immune response proteins recognize foreign nucleic acids from invading pathogens to initiate antiviral signaling. These proteins mostly rely on structural characteristics of the nucleic acids rather than their specific sequences to distinguish self and nonself. One feature utilized by RNA sensors is the extended stretch of double‐stranded RNA (dsRNA) base pairs. However, the criteria for recognizing nonself dsRNAs are rather lenient, and hairpin structure of self‐RNAs can also trigger an immune response. Consequently, aberrant activation of RNA sensors has (...) been reported in numerous human diseases. Yet, in most cases, the activating antigens remain unknown. Recent studies have developed sequencing techniques tailored to specifically capture dsRNAs and identified that various noncoding elements in the nuclear and the mitochondrial genome can generate dsRNAs. Here, the identity of endogenous dsRNAs, their recognition by dsRNA sensors, and their implications in the pathogenesis of human diseases ranging from inflammatory to degenerative are presented. (shrink)

Transcriptome sequencing has identified more than a thousand potentially imprinted genes in the mouse brain. This comes as a revelation to someone who cut his teeth on the identification of imprinted genes when only a handful was known. Genomic imprinting, an epigenetic mechanism that determines expression of alleles according to sex of transmitting parent, was discovered over 25 years ago in mice but remains an enigmatic phenomenon. Why do these genes disobey the normal Mendelian logic of inheritance, do they (...) function in specific processes, and how is their imprinting conferred? Next generation sequencing technologies are providing an unprecedented opportunity to survey the whole genome for imprinted genes and are beginning to reveal that imprinting may be more pervasive than we had come to believe. Such advances should lay the foundation for a definitive account of imprinting, but may also challenge accepted views on what it means to be imprinted.Editor's suggested further reading in BioEssays RNA as the substrate for epigenome‐environment interactions Abstract. (shrink)

The Alu sequence is a SINE (Short INterspersed Element) that is abundant in the human genome. A new analysis(1) reveals an unexpected conservation of some bases in the DNA sequence of the element. The bases involved include those forming an RNA polymerase III promoter. An unresolved question is whether this conservation results from selection for transposability. This, in turn, is related to the larger question of the evolutionary relationship between members of the Alu sequence family.