RESUMEN
Membraneless organelles (MLOs) are detected in cells as dots of mesoscopic size. By undergoing phase separation into a liquid-like or gel-like phase, MLOs contribute to intracellular compartmentalization of specific biological functions. In eukaryotes, dozens of MLOs have been identified, including the nucleolus, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukaemia protein (PML) nuclear bodies, nuclear stress bodies, processing bodies (P bodies) and stress granules. MLOs contain specific proteins, of which many possess intrinsically disordered regions (IDRs), and nucleic acids, mainly RNA. Many MLOs contribute to gene regulation by different mechanisms. Through sequestration of specific factors, MLOs promote biochemical reactions by simultaneously concentrating substrates and enzymes, and/or suppressing the activity of the sequestered factors elsewhere in the cell. Other MLOs construct inter-chromosomal hubs by associating with multiple loci, thereby contributing to the biogenesis of macromolecular machineries essential for gene expression, such as ribosomes and spliceosomes. The organization of many MLOs includes layers, which might have different biophysical properties and functions. MLOs are functionally interconnected and are involved in various diseases, prompting the emergence of therapeutics targeting them. In this Review, we introduce MLOs that are relevant to gene regulation and discuss their assembly, internal structure, gene-regulatory roles in transcription, RNA processing and translation, particularly in stress conditions, and their disease relevance.
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Condensados Biomoleculares , Orgánulos , Orgánulos/metabolismo , ARN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismoRESUMEN
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.
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ARN Largo no Codificante , ARN Largo no Codificante/genética , Núcleo Celular/genética , Cromatina/genética , Secuencias Reguladoras de Ácidos Nucleicos , ARN Polimerasa II/genéticaRESUMEN
4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.
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Empalme del ARN , ARN Pequeño no Traducido , Ratones , Animales , Empalme del ARN/genética , Exones/genética , Retroelementos/genética , Codón sin Sentido , Empalme AlternativoRESUMEN
Power analysis currently dominates sample size determination for experiments, particularly in grant and ethics applications. Yet, this focus could paradoxically result in suboptimal study design because publication biases towards studies with the largest effects can lead to the overestimation of effect sizes. In this Essay, we propose a paradigm shift towards better study designs that focus less on statistical power. We also advocate for (pre)registration and obligatory reporting of all results (regardless of statistical significance), better facilitation of team science and multi-institutional collaboration that incorporates heterogenization, and the use of prospective and living meta-analyses to generate generalizable results. Such changes could make science more effective and, potentially, more equitable, helping to cultivate better collaborations.
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Proyectos de Investigación , Estudios Prospectivos , Tamaño de la Muestra , Sesgo de PublicaciónRESUMEN
A recent article claimed that researchers need not increase the overall sample size for a study that includes both sexes. This Formal Comment points out that that study assumed two sexes to have the same variance, and explains why this is a unrealistic assumption.
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Proyectos de Investigación , Masculino , Femenino , Humanos , Tamaño de la MuestraRESUMEN
Awards can propel academic careers. They also reflect the culture and values of the scientific community. But do awards incentivize greater transparency, inclusivity, and openness in science? Our cross-disciplinary survey of 222 awards for the "best" journal articles across all 27 SCImago subject areas revealed that journals and learned societies administering such awards generally publish little detail on their procedures and criteria. Award descriptions were brief, rarely including contact details or information on the nominations pool. Nominations of underrepresented groups were not explicitly encouraged, and concepts that align with Open Science were almost absent from the assessment criteria. At the same time, 10% of awards, especially the recently established ones, tended to use article-level impact metrics. USA-affiliated researchers dominated the winner's pool (48%), while researchers from the Global South were uncommon (11%). Sixty-one percent of individual winners were men. Overall, Best Paper awards miss the global calls for greater transparency and equitable access to academic recognition. We provide concrete and implementable recommendations for scientific awards to improve the scientific recognition system and incentives for better scientific practice.
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Distinciones y Premios , Humanos , Investigadores , Masculino , Femenino , Ciencia , Edición/normas , Publicaciones Periódicas como Asunto/normasRESUMEN
To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli.
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Paraspeckles , ARN Largo no Codificante , Humanos , Animales , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Macrófagos/metabolismoRESUMEN
Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 are not yet fully understood. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that coregulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.
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Adipocitos Beige , Diferenciación Celular , Frío , Ratones Noqueados , ARN Largo no Codificante , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratones , Diferenciación Celular/genética , Adipocitos Beige/metabolismo , Adipocitos Beige/citología , Termogénesis/genéticaRESUMEN
A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.
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ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estabilidad del ARN , Factores de Transcripción/metabolismoRESUMEN
Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs. Their characteristic cylindrical shapes, with highly ordered internal organization, distinguish them from typical liquid-liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. Using the soft matter physics, we then applied a theoretical framework to understand the principles that determine NEAT1_2 organization as well as shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through a type of microphase separation, micellization. This work provides an experiment-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding biomolecular condensates with optimal sizes and structures in cells.
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Micelas , Polímeros , ARN Largo no Codificante , Ribonucleoproteínas , Línea Celular , HumanosRESUMEN
The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1-/- mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in KrasG12D-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.
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Transformación Celular Neoplásica/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Carcinoma Ductal Pancreático/fisiopatología , Células Cultivadas , Reparación del ADN/genética , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Humanos , RatonesRESUMEN
The rapid urbanization of our world has led to a surge in artificial lighting at night (ALAN), with profound effects on wildlife. Previous research on wildlife's melatonin, a crucial mechanistic indicator and mediator, has yielded inconclusive evidence due to a lack of comparative analysis. We compiled and analysed an evidence base including 127 experiments with 437 observations across 31 wild vertebrates using phylogenetically controlled multilevel meta-analytic models. The evidence comes mainly from the effects of white light on melatonin suppression in birds and mammals. We show a 36% average decrease in melatonin secretion in response to ALAN across a diverse range of species. This effect was observed for central and peripheral melatonin, diurnal and nocturnal species, and captive and free-living populations. We also reveal intensity-, wavelength-, and timing-dependent patterns of ALAN effects. Exposure to ALAN led to a 23% rise in inter-individual variability in melatonin suppression, with important implications for natural selection in wild vertebrates, as some individuals may display higher tolerance to ALAN. The cross-species evidence has strong implications for conservation of wild populations that are subject to natural selection of ALAN. We recommend measures to mitigate harmful impacts of ALAN, such as using 'smart' lighting systems to tune the spectra to less harmful compositions.
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Melatonina , Humanos , Animales , Contaminación Lumínica , Luz , Iluminación , Animales Salvajes , MamíferosRESUMEN
In a growing digital landscape, enhancing the discoverability and resonance of scientific articles is essential. Here, we offer 10 recommendations to amplify the discoverability of studies in search engines and databases. Particularly, we argue that the strategic use and placement of key terms in the title, abstract and keyword sections can boost indexing and appeal. By surveying 230 journals in ecology and evolutionary biology, we found that current author guidelines may unintentionally limit article findability. Our survey of 5323 studies revealed that authors frequently exhaust abstract word limits-particularly those capped under 250 words. This suggests that current guidelines may be overly restrictive and not optimized to increase the dissemination and discoverability of digital publications. Additionally, 92% of studies used redundant keywords in the title or abstract, undermining optimal indexing in databases. We encourage adopting structured abstracts to maximize the incorporation of key terms in titles, abstracts and keywords. In addition, we encourage the relaxation of abstract and keyword limitations in journals with strict guidelines, and the inclusion of multilingual abstracts to broaden global accessibility. These recommendations to editors are designed to improve article engagement and facilitate evidence synthesis, thereby aligning scientific publishing with the modern needs of academic research.
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Publicaciones Periódicas como Asunto , Ecología/métodos , Indización y Redacción de Resúmenes , Edición/normasRESUMEN
A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of "nondomain biomolecules," whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.
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Proteínas , Proteínas/genética , Secuencia Conservada , BiopolímerosRESUMEN
Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
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Paraspeckles , ARN Largo no Codificante , Animales , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratas Topo/genética , Ratas Topo/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.
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Cambio Climático , Invertebrados , Animales , Temperatura , Fertilidad , DrosophilaRESUMEN
The replicability of research results has been a cause of increasing concern to the scientific community. The long-held belief that experimental standardization begets replicability has also been recently challenged, with the observation that the reduction of variability within studies can lead to idiosyncratic, lab-specific results that cannot be replicated. An alternative approach is to, instead, deliberately introduce heterogeneity, known as "heterogenization" of experimental design. Here, we explore a novel perspective in the heterogenization program in a meta-analysis of variability in observed phenotypic outcomes in both control and experimental animal models of ischemic stroke. First, by quantifying interindividual variability across control groups, we illustrate that the amount of heterogeneity in disease state (infarct volume) differs according to methodological approach, for example, in disease induction methods and disease models. We argue that such methods may improve replicability by creating diverse and representative distribution of baseline disease state in the reference group, against which treatment efficacy is assessed. Second, we illustrate how meta-analysis can be used to simultaneously assess efficacy and stability (i.e., mean effect and among-individual variability). We identify treatments that have efficacy and are generalizable to the population level (i.e., low interindividual variability), as well as those where there is high interindividual variability in response; for these, latter treatments translation to a clinical setting may require nuance. We argue that by embracing rather than seeking to minimize variability in phenotypic outcomes, we can motivate the shift toward heterogenization and improve both the replicability and generalizability of preclinical research.
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Experimentación Animal/normas , Proyectos de Investigación/normas , Animales , Conducta Animal/fisiología , Isquemia Encefálica/metabolismo , Humanos , Metaanálisis como Asunto , Modelos Animales , Fenotipo , Estándares de Referencia , Reproducibilidad de los Resultados , Proyectos de Investigación/tendencias , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Selective processes act on phenotypic variation although the evolutionary potential of a trait relies on the underlying heritable variation. Developmental plasticity is an important source of phenotypic variation, but it can also promote changes in genetic variation, yet we have a limited understanding of how they are both impacted. Here, we quantified the influence of developmental temperature on growth in delicate skinks (Lampropholis delicata) and partitioned total phenotypic variance using an animal model fitted with a genomic relatedness matrix. We measured mass for 261 individuals (nhot = 125, ncold = 136) over 16 months (nobservations = 3002) and estimated heritability and maternal effects over time. Our results show that lizards reared in cold developmental temperatures had consistently higher mass across development compared to lizards that were reared in hot developmental temperatures. However, developmental temperature did not impact the rate of growth. On average, additive genetic variance, maternal effects and heritability were higher in the hot developmental temperature treatment; however, these differences were not statistically significant. Heritability increased with age, whereas maternal effects decreased upon hatching but increased again at a later age, which could be driven by social competition or intrinsic changes in the expression of variation as an individual's growth. Our work suggests that the evolutionary potential of growth is complex, age-dependent and not overtly affected by extremes in natural nest temperatures.
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Lagartos , Oviparidad , Animales , Lagartos/genética , Temperatura , Calor , Evolución BiológicaRESUMEN
Meta-analysis is the quantitative, scientific synthesis of research results. Since the term and modern approaches to research synthesis were first introduced in the 1970s, meta-analysis has had a revolutionary effect in many scientific fields, helping to establish evidence-based practice and to resolve seemingly contradictory research outcomes. At the same time, its implementation has engendered criticism and controversy, in some cases general and others specific to particular disciplines. Here we take the opportunity provided by the recent fortieth anniversary of meta-analysis to reflect on the accomplishments, limitations, recent advances and directions for future developments in the field of research synthesis.
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Disciplinas de las Ciencias Biológicas/métodos , Metaanálisis como Asunto , Investigación , Animales , Evolución Biológica , Ecología/métodos , Historia del Siglo XX , Historia del Siglo XXI , Investigación/historia , Investigación/tendenciasRESUMEN
A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.