RESUMO
The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estresse Fisiológico , Ubiquitinas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Condensados Biomoleculares/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Concentração de Íons de Hidrogênio , Grânulos de Estresse/metabolismoRESUMO
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Assuntos
Nucléolo Celular , Proteínas Nucleares , Força Próton-Motriz , Nucléolo Celular/química , Núcleo Celular/química , Proteínas Nucleares/química , RNA/metabolismo , Separação de Fases , Proteínas Intrinsicamente Desordenadas/química , Animais , Xenopus laevis , Oócitos/química , Oócitos/citologiaRESUMO
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
Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Polimerase I/metabolismo , Precursores de RNA/genética , RNA Longo não Codificante/metabolismo , Sítio Alostérico , Animais , Carcinogênese , Linhagem Celular , Linhagem Celular Tumoral , RNA Helicases DEAD-box/química , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Nus , Precursores de RNA/metabolismo , Transcrição GênicaRESUMO
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and â¼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
Assuntos
Chaetomium/química , Biogênese de Organelas , Subunidades Ribossômicas Maiores de Eucariotos/química , Chaetomium/citologia , Microscopia Crioeletrônica , Redes e Vias Metabólicas , Modelos Moleculares , Dobramento de RNA , Ribonucleoproteínas/químicaRESUMO
Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.
Assuntos
Neoplasias , Ribossomos , Camundongos , Animais , Ribossomos/metabolismo , Envelhecimento/genética , Peptídeos/metabolismo , Sirolimo/farmacologia , Neoplasias/metabolismo , Nucléolo Celular/genética , MamíferosRESUMO
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72 , Nucléolo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Expansão das Repetições de DNA , Dipeptídeos/genética , Drosophila melanogaster/genética , Demência Frontotemporal/genética , Humanos , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/genéticaRESUMO
The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (â¼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.
Assuntos
RNA Ribossômico , RNA , RNA/genética , RNA Ribossômico/genética , Condensados Biomoleculares , Nucléolo Celular/genética , Proteínas Nucleares/genéticaRESUMO
DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.
Assuntos
RNA Helicases DEAD-box , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/química , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Organelas/metabolismo , Concentração de Íons de HidrogênioRESUMO
The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.
Assuntos
Cromatina , Transcrição Gênica , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that basket formation is dependent on RNA polymerase II transcription and subsequent mRNP processing. We further show that while all NPCs can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs, and these basket-containing NPCs associate a distinct protein and RNA interactome. Taken together, our data point toward NPC heterogeneity and an RNA-dependent mechanism for functionalization of NPCs in budding yeast through nuclear basket assembly.
Assuntos
Poro Nuclear , Saccharomycetales , Poro Nuclear/genética , Poro Nuclear/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteômica , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. Here we describe a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. As a proof of principle, we show that HyPro-MS and HyPro-seq can identify both known and previously unexplored spatial neighbors of the noncoding RNAs 45S, NEAT1, and PNCTR expressed at markedly different levels. Notably, HyPro-seq uncovers an extensive repertoire of incompletely processed, adenosine-to-inosine-edited transcripts accumulating at the interface between their encoding chromosomal regions and the NEAT1-containing paraspeckle compartment. At least some of these targets require NEAT1 for their optimal expression. Overall, this study provides a versatile toolkit for dissecting RNA interactomes in diverse biomedical contexts and expands our understanding of the functional architecture of the mammalian nucleus.
Assuntos
Compartimento Celular , Núcleo Celular/metabolismo , Técnicas Genéticas , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Espectrometria de Massas , Estudo de Prova de Conceito , Ligação Proteica , Proteoma , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Nuclear/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , TranscriptomaRESUMO
We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Cromossomos , DNA/metabolismo , Linfoma de Células B/metabolismo , Proteínas Nucleares/metabolismo , Prófase , Proteoma , Proteômica , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Galinhas , Cromatina/genética , DNA/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas Nucleares/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fatores de TempoRESUMO
Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.
Assuntos
Sarcoma de Ewing , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Mamíferos/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Ativação Transcricional/genéticaRESUMO
The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are thought to cycle between two states: an operative state (when integrated into subunit assembly intermediates) and a latent state (upon release from intermediates). Although it has become commonplace to refer to the nucleolus as "being a multilayered condensate," and this may be accurate for latent factors, there is little reason to think that such assertions pertain to the operative state of assembly factors.
Assuntos
Nucléolo Celular , RNA RibossômicoRESUMO
Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes â¼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.
Assuntos
RNA Polimerase II , Precursores de RNA , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Intergênico , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Homeostase/genética , Substâncias Macromoleculares/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Transcrição GênicaRESUMO
Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.
Assuntos
Nucléolo Celular , Embrião de Mamíferos , Animais , Nucléolo Celular/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias , Genoma , Mamíferos/genética , Camundongos , RNA Ribossômico/genéticaRESUMO
It is unknown how ribosomal gene (rDNA) arrays from multiple chromosomal nucleolar organizers (NORs) partition within human nucleoli. Exploration of this paradigm for chromosomal organization is complicated by the shared DNA sequence composition of five NOR-bearing acrocentric chromosome p-arms. Here, we devise a methodology for genetic manipulation of individual NORs. Efficient "scarless" genome editing of rDNA repeats is achieved on "poised" human NORs held within monochromosomal cell hybrids. Subsequent transfer to human cells introduces "active" NORs yielding readily discernible functional customized ribosomes. We reveal that ribosome biogenesis occurs entirely within constrained territories, tethered to individual NORs inside a larger nucleolus.
Assuntos
Nucléolo Celular/metabolismo , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Linhagem Celular , Nucléolo Celular/genética , Cromossomos/metabolismo , Edição de Genes , Humanos , Ribossomos/genéticaRESUMO
The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.
Assuntos
Proteínas de Drosophila , Ecdisona , Fatores de Transcrição , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ecdisona/metabolismo , Larva/metabolismo , Larva/crescimento & desenvolvimento , Larva/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Membrana Nuclear/metabolismo , Membrana Nuclear/genética , Drosophila/metabolismo , Drosophila/genética , Cromatina/metabolismo , Nucléolo Celular/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/genética , Mutação/genética , Proteínas RepressorasRESUMO
Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.