RESUMO
Biomolecular condensates appear throughout the cell, serving many different biochemical functions. We argue that condensate functionality is optimized when the interactions driving condensation vary widely in affinity. Strong interactions provide structural specificity needed to encode functional properties but carry the risk of kinetic arrest, while weak interactions allow the system to remain dynamic but do not restrict the conformational ensemble enough to sustain specific functional features. To support our opinion, we describe illustrative examples of the interplay of strong and weak interactions that are found in the nucleolus, SPOP/DAXX condensates, polySUMO/polySIM condensates, chromatin, and stress granules. The common feature of these systems is a hierarchical assembly motif in which weak, transient interactions condense structurally defined functional units.
Assuntos
Carrapatos , Animais , Cromatina , Cinética , OrganelasRESUMO
The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.
Assuntos
Nucléolo Celular , Animais , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/química , Microscopia , Ribossomos/metabolismo , Ribossomos/químicaRESUMO
Proteins and RNAs inside the cell nucleus are organized into distinct phases, also known as liquid-liquid phase separated (LLPS) droplet organelles or nuclear bodies. These regions exist within the spaces between chromatin-rich regions but their function is tightly linked to gene activity. They include major microscopically-observable structures such as the nucleolus, paraspeckle and Cajal body. The biochemical and assembly factors enriched inside these microenvironments regulate chromatin structure, transcription, and RNA processing, and other important cellular functions. Here, we describe published evidence that suggests nuclear bodies are bona fide LLPS droplet organelles and major regulators of the processes listed above. We also outline an updated "Supply or Sequester" model to describe nuclear body function, in which proteins or RNAs are supplied to surrounding genomic regions or sequestered away from their sites of activity. Finally, we describe recent evidence that suggests these microenvironments are both reflective and drivers of diverse pathophysiological states.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Núcleo Celular/química , Separação Celular , Epigênese Genética/genética , Extração Líquido-Líquido , Organelas/metabolismo , RNA/metabolismo , Núcleo Celular/metabolismo , Estruturas do Núcleo Celular/química , Estruturas do Núcleo Celular/genética , Humanos , Organelas/química , Organelas/genética , Tamanho da Partícula , RNA/genética , RNA/isolamento & purificaçãoRESUMO
Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3'-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a ß-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3'-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator 'cleavage module' responsible for its endonucleolytic activity.
Assuntos
Endorribonucleases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Drosophila/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/química , RNA Nuclear Pequeno/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The linker histone H1 is a key player in chromatin organization, yet our understanding of the regulation of H1 functions by post-translational modifications is very limited. We provide here the first functional characterization of H1 acetylation. We show that H1.4K34 acetylation (H1.4K34ac) is mediated by GCN5 and is preferentially enriched at promoters of active genes, where it stimulates transcription by increasing H1 mobility and recruiting a general transcription factor. H1.4K34ac is dynamic during spermatogenesis and marks undifferentiated cells such as induced pluripotent stem (iPS) cells and testicular germ cell tumors. We propose a model for H1.4K34ac as a novel regulator of chromatin function with a dual role in transcriptional activation.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases , Histonas/genética , Humanos , Lisina/genética , Masculino , Dados de Sequência Molecular , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Seminoma/genética , Seminoma/metabolismo , Espermatogênese/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Fator de Transcrição TFIID/metabolismo , Sítio de Iniciação de Transcrição , Regulação para CimaRESUMO
An intrinsic and essential trait exhibited by cells is the properly coordinated and integrated regulation of an astoundingly large number of simultaneous molecular decisions and reactions to maintain biochemical homeostasis. This is especially true inside the cell nucleus, where the recognition of DNA and RNA by a vast range of nucleic acid-interacting proteins organizes gene expression patterns. However, this dynamic system is not regulated by simple "on" or "off" signals. Instead, transcription factor and RNA polymerase recruitment to DNA are influenced by the local chromatin and epigenetic environment, a gene's relative position within the nucleus and the action of noncoding RNAs. In addition, major phase-separated structural features of the nucleus, such as nucleoli and paraspeckles, assemble in direct response to specific transcriptional activities and, in turn, influence global genomic function. Currently, the interpretation of these data is trapped in a causality dilemma reminiscent of the "chicken and the egg" paradox as it is unclear whether changes in nuclear architecture promote RNA function or vice versa. Here, we review recent advances that suggest a complex and interdependent interaction network between gene expression, chromatin topology, and noncoding RNA function. We also discuss the functional links between these essential nuclear processes from the nanoscale (gene looping) to the macroscale (sub-nuclear gene positioning and nuclear body function) and briefly highlight some of the challenges that researchers may encounter when studying these phenomena.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/metabolismo , Animais , DNA/metabolismo , Epigênese Genética , Humanos , Análise EspacialRESUMO
Nuclear bodies contribute to non-random organization of the human genome and nuclear function. Using a major prototypical nuclear body, the Cajal body, as an example, we suggest that these structures assemble at specific gene loci located across the genome as a result of high transcriptional activity. Subsequently, target genes are physically clustered in close proximity in Cajal body-containing cells. However, Cajal bodies are observed in only a limited number of human cell types, including neuronal and cancer cells. Ultimately, Cajal body depletion perturbs splicing kinetics by reducing target small nuclear RNA (snRNA) transcription and limiting the levels of spliceosomal snRNPs, including their modification and turnover following each round of RNA splicing. As such, Cajal bodies are capable of shaping the chromatin interaction landscape and the transcriptome by influencing spliceosome kinetics. Future studies should concentrate on characterizing the direct influence of Cajal bodies upon snRNA gene transcriptional dynamics. Also see the video abstract here.
Assuntos
Corpos Enovelados/genética , Genoma Humano , Spliceossomos , Transcriptoma , Corpos Enovelados/metabolismo , HumanosRESUMO
The assembly of specialized sub-nuclear microenvironments known as nuclear bodies (NBs) is important for promoting efficient nuclear function. In particular, the Cajal body (CB), a prominent NB that facilitates spliceosomal snRNP biogenesis, assembles in response to genomic cues. Here, we detail the factors that regulate CB assembly and structural maintenance. These include the importance of transcription at nucleating gene loci, the grouping of these genes on human chromosomes 1, 6 and 17, as well as cell cycle and biochemical regulation of CB protein function. We also speculate on the correlation between CB formation and RNA splicing levels in neurons and cancer. The timing and location of these specific molecular events is critical to CB assembly and its contribution to genome function. However, further work is required to explore the emerging biophysical characteristics of CB assembly and the impact upon subsequent genome reorganization.
Assuntos
Corpos Enovelados/metabolismo , Genoma , Genômica , Animais , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Corpos Enovelados/genética , Genômica/métodos , Histonas/genética , Histonas/metabolismo , Humanos , Ligação Proteica , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Transcrição GênicaRESUMO
Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes.
Assuntos
Diferenciação Celular/genética , DNA Ribossômico/genética , Células-Tronco Mesenquimais/citologia , Transativadores/genética , Animais , Células COS , Linhagem da Célula , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Chlorocebus aethiops , Cromatina/genética , DNA Helicases , Regulação da Expressão Gênica , Genes de RNAr , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Ribossomos/genética , Transativadores/metabolismoRESUMO
c-myc and p53 networks control proliferation, differentiation, and apoptosis and are responsive to, and cross-regulate a variety of stresses and metabolic and biosynthetic processes. At c-myc, the far upstream element binding protein (FBP) and FBP-interacting repressor (FIR) program transcription by looping to RNA polymerase II complexes engaged at the promoter. Another FBP partner, JTV1/AIMP2, a structural subunit of a multi-aminoacyl-tRNA synthetase (ARS) complex, has also been reported to stabilize p53 via an apparently independent mechanism. Here, we show that in response to oxidative stress, JTV1 dissociates from the ARS complex, translocates to the nucleus, associates with FBP and co-activates the transcription of a new FBP target, ubiquitin-specific peptidase 29 (USP29). A previously uncharacterized deubiquitinating enzyme, USP29 binds to, cleaves poly-ubiquitin chains from, and stabilizes p53. The accumulated p53 quickly induces apoptosis. Thus, FBP and JTV1 help to coordinate the molecular and cellular response to oxidative stress.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/genética , Estresse Oxidativo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Aminoacil-tRNA Sintetases/genética , Apoptose , Biomarcadores/metabolismo , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Endopeptidases/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Ubiquitina/metabolismo , Proteases Específicas de UbiquitinaRESUMO
UNLABELLED: The establishment of a latent reservoir by human tumor viruses is a vital step in initiating cellular transformation and represents a major shortcoming to current therapeutic strategies and the ability to eradicate virus-infected cells. Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong infection and is linked to adult T-cell leukemia lymphoma (ATLL). Here, we demonstrate that HTLV-1 p30 recruits the cellular proteasome activator PA28γ onto the viral tax/rex mRNA to prevent its nuclear export and suppress virus replication. Interaction of p30 with a PA28γ retaining fully functional proteasome activity is required for p30's ability to repress HTLV-1. Consistently, HTLV-1 molecular clones replicate better and produce more virus particles in PA28γ-deficient cells. These results define a unique and novel role for the cellular factor PA28γ in the control of nuclear RNA trafficking and HTLV-1induced latency. Importantly, knockdown of PA28γ expression in ATLL cells latently infected with HTLV-1 reactivates expression of viral tax/rex RNA and the Tax protein. Because Tax is the most immunogenic viral antigen and triggers strong CTL responses, our results suggest that PA28γ-targeted therapy may reactivate virus expression from latently infected cells and allow their eradication from the host. KEY POINTS: PA28γ acts as a co-repressor of HTLV-1 p30 to suppress virus replication and is required for the maintenance of viral latency. HTLV-1 has evolved a unique function mediated by its posttranscriptional repressor p30, which is not found in HTLV-2.
Assuntos
Autoantígenos/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Autoantígenos/genética , Transporte Biológico Ativo/genética , Linhagem Celular , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Produtos do Gene tax/genética , Produtos do Gene tax/metabolismo , Humanos , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Células HeLa , Heterocromatina/metabolismo , Histonas/química , Histonas/genética , Humanos , Metilação , Camundongos , Mitose/fisiologia , Células NIH 3T3 , Fosforilação , Isoformas de Proteínas , Especificidade por SubstratoRESUMO
Trafficking of RNA molecules and proteins within the cell nucleus is central to genome function. Recent work has revealed the nature of RNA and protein motion within the nucleus and across the nuclear membrane. These studies have given insight into how molecules find their destinations within the nucleus and have uncovered some of the structural properties of the nuclear microenvironment. Control of RNA and protein trafficking is now emerging as a physiological regulatory mechanism in gene expression and nuclear function.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Transporte Proteico , RNA/metabolismo , Animais , Difusão , Humanos , Modelos Biológicos , Transdução de SinaisRESUMO
The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Cromatina/genética , Histonas/genética , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação ProteicaRESUMO
Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.
Assuntos
Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas dos Retroviridae/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Genes Reporter , Genes pX , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas dos Retroviridae/genética , Transcrição GênicaRESUMO
During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/fisiologia , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Substâncias Macromoleculares , Mutagênese , Região Organizadora do Nucléolo/metabolismo , Nucleossomos/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.
Assuntos
Corpos Enovelados/metabolismo , Animais , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Substâncias Macromoleculares , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Spliceossomos/metabolismo , Fatores de TempoRESUMO
The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal alpha-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , DNA/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Proteínas Repressoras/metabolismo , Frações Subcelulares , Transativadores/química , Fatores de TranscriçãoRESUMO
We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.
Assuntos
Nucléolo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de RNA/metabolismo , Ribossomos/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Nucléolo Celular/química , Nucléolo Celular/ultraestrutura , Humanos , Proteínas Nucleares/metabolismo , Inibidores de Proteassoma , Precursores de RNA/análise , Precursores de RNA/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Ribossomos/genética , Transcrição Gênica , Ubiquitina/análiseRESUMO
Cells are segregated into two distinct compartment groups to optimize cellular function. The first is characterized by lipid membranes that encapsulate specific regions and regulate macromolecular flux. The second, known collectively as membraneless organelles (MLOs), lacks defining lipid membranes and exhibits self-organizing properties. MLOs are enriched with specific RNAs and proteins that catalyze essential cellular processes. A prominent sub-class of MLOs are known as nuclear bodies, which includes nucleoli, paraspeckles, and other droplets. These microenvironments contain specific RNAs, exhibit archetypal liquid-liquid phase separation characteristics, and harbor intrinsically disordered, multivalent hub proteins. We present an analysis of nuclear body protein disorder that suggests MLO proteomes are significantly more disordered than structured cellular features. We also outline common MLO ultrastructural features, exemplified by the three sub-compartments present inside the nucleolus. A core-shell configuration, or phase within a phase, is displayed by several nuclear bodies and may be functionally important. Finally, we summarize evidence indicating extensive RNA and protein sharing between distinct nuclear bodies, suggesting functional cooperation and similar nucleation principles. Considering the substantial accumulation of specific coding and noncoding RNA classes inside MLOs, evidence that RNA buffers specific phase transition events, and the absence of a clear correlation between total intrinsic protein disorder and MLO accumulation, we conclude that RNA biogenesis may play a key role in MLO formation, internal organization, and function. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.