Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.

Natural antisense transcripts as versatile regulators of gene expression.

Long non-coding RNAs (lncRNAs) are emerging as a major class of gene products that have central roles in cell and developmental biology. Natural antisense transcripts (NATs) are an important subset of lncRNAs that are expressed from the opposite strand of protein-coding and non-coding genes and are a genome-wide phenomenon in both eukaryotes and prokaryotes. In eukaryotes, a myriad of NATs participate in regulatory pathways that affect expression of their cognate sense genes. Recent developments in the study of NATs and lncRNAs and large-scale sequencing and bioinformatics projects suggest that whether NATs regulate expression, splicing, stability or translation of the sense transcript is influenced by the pattern and degrees of overlap between the sense-antisense pair. Moreover, epigenetic gene regulatory mechanisms prevail in somatic cells whereas mechanisms dependent on the formation of double-stranded RNA intermediates are prevalent in germ cells. The modulating effects of NATs on sense transcript expression make NATs rational targets for therapeutic interventions.

Charting the unknown epitranscriptome.

RNA modifications can alter RNA structure-function relationships and various cellular processes. However, the genomic distribution and biological roles of most RNA modifications remain uncharacterized. Here, we propose using phage display antibody technology and direct sequencing through nanopores to facilitate systematic interrogation of the distribution, location and dynamics of RNA modifications.

The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

RNA out of the mist.

RNA has long been regarded primarily as the intermediate between genes and proteins. It was a surprise then to discover that eukaryotic genes are mosaics of mRNA sequences interrupted by large tracts of transcribed but untranslated sequences, and that multicellular organisms also express many long 'intergenic' and antisense noncoding RNAs (lncRNAs). The identification of small RNAs that regulate mRNA translation and half-life did not disturb the prevailing view that animals and plant genomes are full of evolutionary debris and that their development is mainly supervised by transcription factors. Gathering evidence to the contrary involved addressing the low conservation, expression, and genetic visibility of lncRNAs, demonstrating their cell-specific roles in cell and developmental biology, and their association with chromatin-modifying complexes and phase-separated domains. The emerging picture is that most lncRNAs are the products of genetic loci termed 'enhancers', which marshal generic effector proteins to their sites of action to control cell fate decisions during development.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

The human mitochondrial transcriptome.

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

The evolution of ultraconserved elements in vertebrates.

Ultraconserved elements (UCEs) were discovered two decades ago, arbitrarily defined as sequences that are identical over a length ≥200 bp in the human, mouse and rat genomes. The definition was subsequently extended to sequences ≥100 bp identical in at least three of five mammalian genomes (including dog and cow), and shown to have undergone rapid expansion from ancestors in fish and strong negative selection in birds and mammals. Since then, many more genomes have become available, allowing better definition and more thorough examination of UCE distribution and evolutionary history. We developed a fast and flexible analytical pipeline for identifying UCEs in multiple genomes, dedUCE, which allows manipulation of minimum length, sequence identity, and number of species with a detectable UCE according to specified parameters. We suggest an updated definition of UCEs as sequences ≥100 bp and ≥97% sequence identity in ≥50% of placental mammal orders (12813 UCEs). By mapping UCEs to ∼200 species we find that placental UCEs appeared early in vertebrate evolution, well before land colonisation, suggesting the evolutionary pressures driving UCE selection were present in aquatic environments in the Cambrian-Devonian periods. Most (>90%) UCEs likely appeared after the divergence of gnathostomes from jawless predecessors, were largely established in sequence identity by early Sarcopterygii evolution - before the divergence of lobe-finned fishes from tetrapods - and became near fixed in the amniotes. UCEs are mainly located in the introns of protein-coding and non-coding genes involved in neurological and skeletomuscular development, enriched in regulatory elements, and dynamically expressed throughout embryonic development.

A Kuhnian revolution in molecular biology: Most genes in complex organisms express regulatory RNAs.

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.

Widespread formation of double-stranded RNAs in testis.

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes-again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

Exploring the epitranscriptome by native RNA sequencing.

Chemical RNA modifications, collectively referred to as the "epitranscriptome," are essential players in fine-tuning gene expression. Our ability to analyze RNA modifications has improved rapidly in recent years, largely due to the advent of high-throughput sequencing methodologies, which typically consist of coupling modification-specific reagents, such as antibodies or enzymes, to next-generation sequencing. Recently, it also became possible to map RNA modifications directly by sequencing native RNAs using nanopore technologies, which has been applied for the detection of a number of RNA modifications, such as N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). However, the signal modulations caused by most RNA modifications are yet to be determined. A global effort is needed to determine the signatures of the full range of RNA modifications to avoid the technical biases that have so far limited our understanding of the epitranscriptome.

Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

RNA Regulatory Networks 2.0.

The central role of RNA molecules in cell biology has been an expanding subject of study since the proposal of the "RNA world" hypothesis 60 years ago [...].

Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation.

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

Impacts of genomics on the health and social costs of intellectual disability.

BACKGROUND: This study provides an integrated assessment of the economic and social impacts of genomic sequencing for the detection of monogenic disorders resulting in intellectual disability (ID). METHODS: Multiple knowledge bases were cross-referenced and analysed to compile a reference list of monogenic disorders associated with ID. Multiple literature searches were used to quantify the health and social costs for the care of people with ID. Health and social expenditures and the current cost of whole-exome sequencing and whole-genome sequencing were quantified in relation to the more common causes of ID and their impact on lifespan. RESULTS: On average, individuals with ID incur annual costs in terms of health costs, disability support, lost income and other social costs of US$172 000, accumulating to many millions of dollars over a lifetime. CONCLUSION: The diagnosis of monogenic disorders through genomic testing provides the opportunity to improve the diagnosis and management, and to reduce the costs of ID through informed reproductive decisions, reductions in unproductive diagnostic tests and increasingly targeted therapies.

CNS cell type-specific gene profiling of P301S tau transgenic mice identifies genes dysregulated by progressive tau accumulation.

The microtubule-associated protein tau undergoes aberrant modification resulting in insoluble brain deposits in various neurodegenerative diseases, including frontotemporal dementia (FTD), progressive supranuclear palsy, and corticobasal degeneration. Tau aggregates can form in different cell types of the central nervous system (CNS) but are most prevalent in neurons. We have previously recapitulated aspects of human FTD in mouse models by overexpressing mutant human tau in CNS neurons, including a P301S tau variant in TAU58/2 mice, characterized by early-onset and progressive behavioral deficits and FTD-like neuropathology. The molecular mechanisms underlying the functional deficits of TAU58/2 mice remain mostly elusive. Here, we employed functional genomics (i.e. RNAseq) to determine differentially expressed genes in young and aged TAU58/2 mice to identify alterations in cellular processes that may contribute to neuropathy. We identified genes in cortical brain samples differentially regulated between young and old TAU58/2 mice relative to nontransgenic littermates and by comparative analysis with a dataset of CNS cell type-specific genes expressed in nontransgenic mice. Most differentially-regulated genes had known or putative roles in neurons and included presynaptic and excitatory genes. Specifically, we observed changes in presynaptic factors, glutamatergic signaling, and protein scaffolding. Moreover, in the aged mice, expression levels of several genes whose expression was annotated to occur in other brain cell types were altered. Immunoblotting and immunostaining of brain samples from the TAU58/2 mice confirmed altered expression and localization of identified and network-linked proteins. Our results have revealed genes dysregulated by progressive tau accumulation in an FTD mouse model.

The Dimensions, Dynamics, and Relevance of the Mammalian Noncoding Transcriptome.

The combination of pervasive transcription and prolific alternative splicing produces a mammalian transcriptome of great breadth and diversity. The majority of transcribed genomic bases are intronic, antisense, or intergenic to protein-coding genes, yielding a plethora of short and long non-protein-coding regulatory RNAs. Long noncoding RNAs (lncRNAs) share most aspects of their biogenesis, processing, and regulation with mRNAs. However, lncRNAs are typically expressed in more restricted patterns, frequently from enhancers, and exhibit almost universal alternative splicing. These features are consistent with their role as modular epigenetic regulators. We describe here the key studies and technological advances that have shaped our understanding of the dimensions, dynamics, and biological relevance of the mammalian noncoding transcriptome.

The rise of regulatory RNA.

Discoveries over the past decade portend a paradigm shift in molecular biology. Evidence suggests that RNA is not only functional as a messenger between DNA and protein but also involved in the regulation of genome organization and gene expression, which is increasingly elaborate in complex organisms. Regulatory RNA seems to operate at many levels; in particular, it plays an important part in the epigenetic processes that control differentiation and development. These discoveries suggest a central role for RNA in human evolution and ontogeny. Here, we review the emergence of the previously unsuspected world of regulatory RNA from a historical perspective.