RESUMO
How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.
Assuntos
Besouros , Animais , Besouros/genética , Besouros/metabolismo , Evolução Molecular , Benzoquinonas/metabolismo , Filogenia , Genômica , Simbiose/genética , Transcriptoma , Genoma de InsetoRESUMO
RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.
Assuntos
Núcleo Celular/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Homólogo 5 da Proteína Cromobox/metabolismo , Cromossomos/metabolismo , DNA/metabolismo , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dactinomicina/farmacologia , Feminino , Genoma , Células HEK293 , Heterocromatina/metabolismo , Humanos , Camundongos , Modelos Biológicos , Família Multigênica , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The lncRNA Xist forms â¼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain â¼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Longo não Codificante/metabolismo , Cromossomo X/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fibroblastos , Inativação Gênica , Humanos , Camundongos , Ligação Proteica , Inativação do Cromossomo XRESUMO
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.
Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , Núcleo Celular/genética , Cromatina/genética , Sequências Reguladoras de Ácido Nucleico , RNA Polimerase II/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Splicing de RNA , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , COVID-19/virologia , Chlorocebus aethiops , Células HEK293 , Humanos , Interferons/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Citoplasmático Pequeno/química , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
Gene regulation requires the dynamic coordination of hundreds of regulatory factors at precise genomic and RNA targets. Although many regulatory factors have specific affinity for their nucleic acid targets, molecular diffusion and affinity models alone cannot explain many of the quantitative features of gene regulation in the nucleus. One emerging explanation for these quantitative properties is that DNA, RNA and proteins organize within precise, 3D compartments in the nucleus to concentrate groups of functionally related molecules. Recently, nucleic acids and proteins involved in many important nuclear processes have been shown to engage in cooperative interactions, which lead to the formation of condensates that partition the nucleus. In this Review, we discuss an emerging perspective of gene regulation, which moves away from classic models of stoichiometric interactions towards an understanding of how spatial compartmentalization can lead to non-stoichiometric molecular interactions and non-linear regulatory behaviours. We describe key mechanisms of nuclear compartment formation, including emerging roles for non-coding RNAs in facilitating their formation, and discuss the functional role of nuclear compartments in transcription regulation, co-transcriptional and post-transcriptional RNA processing, and higher-order chromatin regulation. More generally, we discuss how compartmentalization may explain important quantitative aspects of gene regulation.
Assuntos
Núcleo Celular/fisiologia , Regulação da Expressão Gênica , Animais , Cromatina/metabolismo , Humanos , Transição de Fase , Processamento Pós-Transcricional do RNA , RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.
Assuntos
Núcleo Celular/ultraestrutura , Mapeamento Cromossômico/métodos , Cromossomos/fisiologia , Nucléolo Celular , Núcleo Celular/fisiologia , Cromossomos/genética , DNA/fisiologia , Eucariotos , Genoma/genética , Genoma/fisiologia , Humanos , Relação Estrutura-AtividadeRESUMO
Exposure to ultraviolet light leads to a cell-wide DNA damage response that includes a global reduction in transcription. Williamson et al., identify a protein involved in this process as well as a noncoding RNA produced by alternative processing of RNA transcribed from the same gene that promotes recovery from the repressed state.
Assuntos
RNA , Transcrição Gênica , Dano ao DNA , RNA não Traduzido/genética , Raios UltravioletaRESUMO
Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.
Assuntos
Complexo Repressor Polycomb 2 , RNA Longo não Codificante , Animais , Camundongos , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromatina/genética , Sítios de LigaçãoRESUMO
Genetic material is not randomly organized within the nucleus of a cell. How this organization occurs and why it matters are questions that Cell editor Marta Koch posed to Mitchell Guttman, Job Dekker, and Stavros Lomvardas. Excerpts from this Conversation are presented below, and an audio file of the full discussion is available with the article online.
Assuntos
Núcleo Celular/química , Cromossomos/química , DNA/química , Animais , Núcleo Celular/genética , Núcleo Celular/fisiologia , Cromossomos/genética , Cromossomos/metabolismo , DNA/genética , DNA/metabolismo , Genômica , National Institutes of Health (U.S.) , Estados UnidosRESUMO
The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.
Assuntos
Genoma , Salpicos Nucleares , Precursores de RNA , Splicing de RNA , RNA Mensageiro , Spliceossomos , Animais , Humanos , Masculino , Camundongos , Genes , Genoma/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Salpicos Nucleares/genética , Salpicos Nucleares/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Transcrição GênicaRESUMO
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
Assuntos
Técnicas Genéticas , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Reagentes de Ligações Cruzadas/metabolismo , Camundongos , Dados de Sequência Molecular , Motivos de Nucleotídeos , Sítios de Splice de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Nuclear Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismoRESUMO
Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.
Assuntos
Pseudouridina/análise , RNA Mensageiro/química , RNA não Traduzido/química , Animais , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Perfilação da Expressão Gênica , Humanos , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pseudouridina/metabolismo , RNA/química , RNA/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Telomerase/química , Telomerase/genéticaRESUMO
Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples - including Xist, which orchestrates X chromosome inactivation - has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus.
Assuntos
Núcleo Celular/ultraestrutura , Regulação da Expressão Gênica , RNA Longo não Codificante/fisiologia , Animais , Núcleo Celular/genética , Expressão Gênica , Humanos , Transporte de RNARESUMO
Large noncoding RNAs are emerging as an important component in cellular regulation. Considerable evidence indicates that these transcripts act directly as functional RNAs rather than through an encoded protein product. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. Here, we show that classical noncoding RNAs and 5' UTRs show the same ribosome occupancy as lincRNAs, demonstrating that ribosome occupancy alone is not sufficient to classify transcripts as coding or noncoding. Instead, we define a metric based on the known property of translation whereby translating ribosomes are released upon encountering a bona fide stop codon. We show that this metric accurately discriminates between protein-coding transcripts and all classes of known noncoding transcripts, including lincRNAs. Taken together, these results argue that the large majority of lincRNAs do not function through encoded proteins.
Assuntos
RNA Longo não Codificante/metabolismo , Ribossomos/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Embrião de Mamíferos/metabolismo , Camundongos , Biossíntese de Proteínas , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA não Traduzido/metabolismoRESUMO
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.
Assuntos
Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Transcrição Gênica , Acetilação , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , Metilação de DNA , Elementos Facilitadores Genéticos , Histonas/metabolismo , Humanos , MetilaçãoRESUMO
Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.
Assuntos
Bactérias/isolamento & purificação , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica , Modelos Lineares , Camundongos Endogâmicos C57BL , Modelos Biológicos , Processos Estocásticos , Fatores de Transcrição/metabolismoRESUMO
Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies1-4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.
Assuntos
Compartimento Celular/genética , Núcleo Celular/genética , Genômica/métodos , Células-Tronco Embrionárias Murinas/citologia , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos de Mamíferos/genética , Células Clonais/citologia , Imunofluorescência , Marcadores Genéticos , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Fatores de TempoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.