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1.
Anal Chem ; 96(24): 9808-9816, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38833718

RESUMO

Visualization of the mitochondrial state is crucial for tracking cell life processes and diagnosing disease, while fluorescent probes that can accurately assess mitochondrial status are currently scarce. Herein, a fluorescent probe named "SYN" was designed and prepared, which can target mitochondria via the mitochondrial membrane potential. Upon pathology or external stimulation, SYN can be released from the mitochondria and accumulate in the nucleolus to monitor the status of mitochondria. During this process, the brightness of the nucleolus can then serve as an indicator of mitochondrial damage. SYN has demonstrated excellent photostability in live cells as well as an extremely inert fluorescence response to bioactive molecules and the physiological pH environment of live cells. Spectroscopic titration and molecular docking studies have revealed that SYN can be lit up in nucleoli due to the high viscosity of the nucleus and the strong electrostatic interaction with the phosphate backbone of RNA. This probe is expected to be an exceptional tool based on its excellent imaging properties for tracking mitochondrial state in live cells.


Assuntos
Nucléolo Celular , Corantes Fluorescentes , Mitocôndrias , Mitocôndrias/metabolismo , Mitocôndrias/química , Humanos , Corantes Fluorescentes/química , Nucléolo Celular/metabolismo , Células HeLa , Simulação de Acoplamento Molecular , Imagem Óptica , Potencial da Membrana Mitocondrial
2.
Nat Commun ; 15(1): 4996, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862527

RESUMO

Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holotomographic microscopy with Artificial Intelligence to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs lipid droplets, changes mitochondrial shape and dry mass, and separates lipid droplets from mitochondria. We then used Bayesian network modeling on organelle dry mass states to define organelle cross-regulation networks and report modifications of organelle cross-regulation that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides an Artificial Intelligence-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.


Assuntos
Teorema de Bayes , COVID-19 , Gotículas Lipídicas , Mitocôndrias , SARS-CoV-2 , SARS-CoV-2/fisiologia , Humanos , COVID-19/virologia , COVID-19/metabolismo , Mitocôndrias/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Inteligência Artificial , Nucléolo Celular/metabolismo , Nucléolo Celular/virologia , Replicação Viral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Animais , Chlorocebus aethiops , Células Vero
3.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38858088

RESUMO

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.


Assuntos
Nucléolo Celular , Proteômica , Ribossomos , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Proteômica/métodos , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Células HeLa , Retículo Endoplasmático/metabolismo
4.
Nanoscale ; 16(24): 11739-11748, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38864270

RESUMO

Ribosomal RNA (rRNA) plays a key role in protein synthesis and ribosomal biogenesis. The exclusively used commercial dye for RNA staining is SYTO RNASelect, which works in fixed cells only. To overcome this constraint, we synthesized NIR-emissive, highly photostable, and biocompatible carbon nanodots (CNDs) as a fluorescent biomarker for rRNA. The synthesized CNDs could stain rRNA in both live and fixed cells. We were able to visualize rRNA at different sites in eukaryotic cells using super-resolution microscopy (SRM). The CNDs localized rRNA in the dense fibrillar components (DFCs) of the nucleolus, nuclear membrane, and rough endoplasmic reticulum (RER). The super-resolved hollow ring-structured DFC with an FWHM of 140 nm, nuclear membrane with an FWHM of 120 nm, and ER with an FWHM of 115 nm were observed. We further found a marked contrast between the pre-RNA synthesized in cancer cells and normal cells. We believe that these CNDs have great potential in rRNA imaging and comprehending the complex relationships between rRNA dynamics and basic biological processes, disease development, or drug interactions.


Assuntos
Carbono , Nucléolo Celular , RNA Ribossômico , Humanos , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Carbono/química , Nucléolo Celular/metabolismo , Pontos Quânticos/química , Microscopia de Fluorescência , Células HeLa , Corantes Fluorescentes/química
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 317: 124434, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38735113

RESUMO

Probing intracellular organelles with fluorescent dyes offers opportunities to understand the structures and functions of these cellular compartments, which is attracting increasing interests. Normally, the design principle varies for different organelle targets as they possess distinct structural and functional profiles against each other. Therefore, developing a probe with dual intracellular targets is of great challenge. In this work, a new sort of donor-π-bridge-acceptor (D-π-A) type coumaranone dyes (CMO-1/2/3/4) have been prepared. Four fluorescent probes (TPP@CMO-1/2/3/4) were then synthesized by linking these coumaranone dyes with an amphiphilic cation triphenylphosphonium (TPP). Interestingly, both TPP@CMO-1 and TPP@CMO-2 exhibited dual color emission upon targeting to two different organelles, respectively. The green emission is well localized in mitochondria, while, the red emission realizes nucleoli imaging. RNA is the target of TPP@CMOs, which was confirmed by spectroscopic analysis and computational calculation. More importantly, the number and morphology changes of nucleoli under drug stress have been successfully evaluated using TPP@CMO-1.


Assuntos
Nucléolo Celular , Corantes Fluorescentes , Mitocôndrias , Compostos Organofosforados , Compostos Organofosforados/química , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Nucléolo Celular/metabolismo , Células HeLa , Espectrometria de Fluorescência , Cor
6.
J Ovarian Res ; 17(1): 99, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38730385

RESUMO

With increasingly used assisted reproductive technology (ART), the acquisition of high-quality oocytes and early embryos has become the focus of much attention. Studies in mice have found that the transition of chromatin conformation from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) is essential for oocyte maturation and early embryo development, and similar chromatin transition also exists in human oocytes. In this study, we collected human NSN and SN oocytes and investigated their transcriptome. The analysis of differentially expressed genes showed that epigenetic functions, cyclin-dependent kinases and transposable elements may play important roles in chromatin transition during human oocyte maturation. Our findings provide new insights into the molecular mechanism of NSN-to-SN transition of human oocyte and obtained new clues for improvement of oocyte in vitro maturation technique.


Assuntos
Cromatina , Oócitos , Transcriptoma , Humanos , Oócitos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Feminino , Perfilação da Expressão Gênica , Nucléolo Celular/metabolismo , Nucléolo Celular/genética
7.
Stem Cell Reports ; 19(5): 689-709, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38701778

RESUMO

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.


Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Animais , Camundongos , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Blastocisto/metabolismo , Blastocisto/citologia , Via de Sinalização Wnt , Proteínas Wnt/metabolismo , Junções Íntimas/metabolismo , Nucléolo Celular/metabolismo
8.
Open Biol ; 14(5): 230358, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38689555

RESUMO

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.


Assuntos
Nucléolo Celular , Cromatina , Desenvolvimento Embrionário , Animais , Humanos , Camundongos , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Zigoto/metabolismo , Zigoto/citologia
9.
Cancer Res Commun ; 4(6): 1495-1504, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38767454

RESUMO

Cancer cells with DNA repair defects (e.g., BRCA1/2 mutant cells) are vulnerable to PARP inhibitors (PARPi) due to induction of synthetic lethality. However, recent clinical evidence has shown that PARPi can prevent the growth of some cancers irrespective of their BRCA1/2 status, suggesting alternative mechanisms of action. We previously discovered one such mechanism in breast cancer involving DDX21, an RNA helicase that localizes to the nucleoli of cells and is a target of PARP1. We have now extended this observation in endometrial and ovarian cancers and provided links to patient outcomes. When PARP1-mediated ADPRylation of DDX21 is inhibited by niraparib, DDX21 is mislocalized to the nucleoplasm resulting in decreased rDNA transcription, which leads to a reduction in ribosome biogenesis, protein translation, and ultimately endometrial and ovarian cancer cell growth. High PARP1 expression was associated with high nucleolar localization of DDX21 in both cancers. High nucleolar DDX21 negatively correlated with calculated IC50s for niraparib. By studying endometrial cancer patient samples, we were able to show that high DDX21 nucleolar localization was significantly associated with decreased survival. Our study suggests that the use of PARPi as a cancer therapeutic can be expanded to further types of cancers and that DDX21 localization can potentially be used as a prognostic factor and as a biomarker for response to PARPi. SIGNIFICANCE: Currently, there are no reliable biomarkers for response to PARPi outside of homologous recombination deficiency. Herein we present a unique potential biomarker, with clear functional understanding of the molecular mechanism by which DDX21 nucleolar localization can predict response to PARPi.


Assuntos
Nucléolo Celular , RNA Helicases DEAD-box , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Feminino , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Prognóstico , Proliferação de Células/efeitos dos fármacos , Neoplasias dos Genitais Femininos/genética , Neoplasias dos Genitais Femininos/patologia , Neoplasias dos Genitais Femininos/tratamento farmacológico , Neoplasias dos Genitais Femininos/mortalidade , Neoplasias dos Genitais Femininos/metabolismo , Indazóis
10.
Nat Cell Biol ; 26(6): 946-961, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38745030

RESUMO

RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.


Assuntos
Proteínas Cromossômicas não Histona , Leucemia Mieloide Aguda , Precursores de RNA , Processamento Pós-Transcricional do RNA , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Animais , Linhagem Celular Tumoral , Biossíntese de Proteínas , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Camundongos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Regulação Leucêmica da Expressão Gênica , Separação de Fases
11.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38717338

RESUMO

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.


Assuntos
DNA Helicases , Enzimas Multifuncionais , RNA Helicases , RNA não Traduzido , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Agregados Proteicos , Proteostase , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Helicases/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
12.
Biochim Biophys Acta Gene Regul Mech ; 1867(2): 195029, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38642633

RESUMO

The perception that the nucleoli are merely the organelles where ribosome biogenesis occurs is challenged. Only around 30 % of nucleolar proteins are solely involved in producing ribosomes. Instead, the nucleolus plays a critical role in controlling protein trafficking during stress and, according to its dynamic nature, undergoes continuous protein exchange with nucleoplasm under various cellular stressors. Hence, the concept of nucleolar stress has evolved as cellular insults that disrupt the structure and function of the nucleolus. Considering the emerging role of this organelle in DNA repair and the fact that rDNAs are the most fragile genomic loci, therapies targeting the nucleoli are increasingly being developed. Besides, drugs that target ribosome synthesis and induce nucleolar stress can be used in cancer therapy. In contrast, agents that regulate nucleolar activity may be a potential treatment for neurodegeneration caused by abnormal protein accumulation in the nucleolus. Here, I explore the roles of nucleoli beyond their ribosomal functions, highlighting the factors triggering nucleolar stress and their impact on genomic stability.


Assuntos
Nucléolo Celular , Instabilidade Genômica , Estresse Fisiológico , Nucléolo Celular/metabolismo , Humanos , Ribossomos/metabolismo , Animais , Reparo do DNA , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , DNA Ribossômico/metabolismo , DNA Ribossômico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
13.
Biomolecules ; 14(4)2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38672417

RESUMO

Silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene and a consequent lack of FMR protein (FMRP) synthesis are associated with fragile X syndrome, one of the most common inherited intellectual disabilities. FMRP is a multifunctional protein that is involved in many cellular functions in almost all subcellular compartments under both normal and cellular stress conditions in neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and it is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes the recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.


Assuntos
Nucléolo Celular , Citosol , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Humanos , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Nucléolo Celular/metabolismo , Citosol/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/genética , Animais , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Processamento de Proteína Pós-Traducional
14.
Biochem Biophys Res Commun ; 714: 149970, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38663097

RESUMO

Movement dynamics in the nucleus involve various biological processes, including DNA repair, which is crucial for cancer prevention. Changes in the movement of the components of the nucleus indicate the changes in movement dynamics in the nucleus. In Schizosaccharomyces pombe, the inner nuclear membrane protein Bqt4 plays an essential role in attaching telomeres to the nuclear envelope. We observed that the deletion of bqt4+ caused a significant decrease in the mean square displacement (MSD) calculated from the distance between the nucleolar center and spindle pole body (SPB), hereafter referred to as MSD(SPB-Nucleolus). The MSD(SPB-Nucleolus) decrease in bqt4Δ was microtubule-dependent. The Rap1-binding ability loss mutant, bqt4F46A, and nonspecific DNA-binding ability mutants, bqt43E-A, did not exhibit an MSD(SPB-Nucleolus) decrease compared to the WT. Moreover, the bqt43E-Arap1Δ double mutant and 1-262 amino acids truncated mutant bqt4ΔN (263-432), which does not have either Rap1-binding or nonspecific DNA-binding abilities, did not exhibit the MSD(SPB-Nucleolus) decrease to the same extent as bqt4Δ. These results suggest that the unknown function of Bqt4 in the C-terminal domain is essential for the maintenance of the pattern of relative movement between SPB and the nucleolus.


Assuntos
Nucléolo Celular , Proteínas de Ligação a DNA , Proteínas Nucleares , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Corpos Polares do Fuso , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Nucléolo Celular/metabolismo , Corpos Polares do Fuso/metabolismo , Mutação , Microtúbulos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ligação Proteica
15.
Mol Cell ; 84(8): 1400-1402, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38640892

RESUMO

Nucleolar stress has been consistently linked to age-related diseases. In this issue, Sirozh et al.1 find that the common molecular signature of nucleolar stress is the accumulation of free ribosomal proteins, which leads to premature aging in mice; however, it can be reversed by mTOR inhibition.


Assuntos
Nucléolo Celular , Proteínas Ribossômicas , Camundongos , Animais , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , RNA Ribossômico/metabolismo
16.
Prostate ; 84(10): 967-976, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38632701

RESUMO

BACKGROUND: Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation. METHODS: Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays. RESULTS: In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis. CONCLUSIONS: Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.


Assuntos
Fator 3-alfa Nuclear de Hepatócito , Neoplasias da Próstata , RNA Ribossômico , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/biossíntese , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Linhagem Celular Tumoral , Transcrição Gênica , Regulação Neoplásica da Expressão Gênica , Nucléolo Celular/metabolismo
17.
EMBO Rep ; 25(6): 2786-2811, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38654122

RESUMO

Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension human cells. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images. Holograms containing cell optical thickness information allowed us to define a novel index which we used to distinguish nucleoli whose material state had been modulated optogenetically by blue-light-induced protein aggregation. Nucleoli whose function had been impacted by drug treatment or depletion of ribosomal proteins could also be distinguished. We explored the potential of the technology to detect other natural and pathological condensates, such as those formed upon overexpression of a mutant form of huntingtin, ataxin-3, or TDP-43, and also other cell assemblies (lipid droplets). We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli and other cell assemblies, including their material state, without any staining.


Assuntos
Nucléolo Celular , Holografia , Humanos , Nucléolo Celular/metabolismo , Holografia/métodos , Redes Neurais de Computação , Microscopia/métodos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Ataxina-3/metabolismo , Ataxina-3/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Microscopia de Contraste de Fase/métodos , Imageamento Quantitativo de Fase
18.
Int J Mol Sci ; 25(7)2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38612434

RESUMO

At the time of diagnosis, Alzheimer's disease (AD) patients already suffer from significant neuronal loss. The identification of proteins that influence disease progression before the onset of symptoms is thus an essential part of the development of new effective drugs and biomarkers. Here, we used an unbiased 18O labelling proteomics approach to identify proteins showing altered levels in the AD brain. We studied the relationship between the protein with the highest increase in hippocampus, DEAD box Helicase 24 (DDX24), and AD pathology. We visualised DDX24 in the human brain and in a mouse model for Aß42-induced AD pathology-AppNL-F-and studied the interaction between Aß and DDX24 in primary neurons. Immunohistochemistry in the AD brain confirmed the increased levels and indicated an altered subcellular distribution of DDX24. Immunohistochemical studies in AppNL-F mice showed that the increase of DDX24 starts before amyloid pathology or memory impairment is observed. Immunocytochemistry in AppNL-F primary hippocampal neurons showed increased DDX24 intensity in the soma, nucleus and nucleolus. Furthermore, siRNA targeting of DDX24 in neurons decreased APP and Aß42 levels, and the addition of Aß42 to the medium reduced DDX24. In conclusion, we have identified DDX24 as a protein with a potential role in Aß-induced AD pathology.


Assuntos
Doença de Alzheimer , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Proteínas Amiloidogênicas , Encéfalo , Nucléolo Celular , RNA Helicases DEAD-box/genética
19.
J Alzheimers Dis ; 98(3): 837-857, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38489184

RESUMO

A hypothesis of Alzheimer's disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli. Polyamines are essential in nucleolar functions, such as RNA folding and ribonucleoprotein assembly. Changes in the nucleolar pool of anionic RNA and cationic polyamines acting as counterions can cause significant nucleolar dynamics. Polyamine synthesis reduces S-adenosylmethionine which, at low levels, triggers tau phosphorylation. Also, polyamine recycling reduces acetyl-CoA needed for acetylcholine, which is low in Alzheimer's disease. Extraordinary nucleolar expansion and/or contraction can disrupt epigenetic control in peri-nucleolar chromatin, such as chromosome 14 with the presenilin-1 gene; chromosome 21 with the amyloid precursor protein gene; chromosome 17 with the tau gene; chromosome 19 with the APOE4 gene; and the inactive X chromosome (Xi; aka "nucleolar satellite") with normally silent spermine synthase (polyamine synthesis) and spermidine/spermine-N1-acetyltransferase (polyamine recycling) alleles. Chromosomes 17, 19 and the Xi have high concentrations of Alu elements which can be transcribed by RNA polymerase III if positioned nucleosomes are displaced from the Alu elements. A sudden flood of Alu RNA transcripts can competitively bind nucleolin which is usually bound to Alu sequences in structural RNAs that stabilize the nucleolar heterochromatic shell. This Alu competition leads to loss of nucleolar integrity with leaking of nucleolar polyamines that cause aggregation of phosphorylated tau. The hypothesis was developed with key word searches (e.g., PubMed) using relevant terms (e.g., Alzheimer's, lupus, nucleolin) based on a systems biology approach and exploring autoimmune disease tautology, gaining synergistic insights from other diseases.


Assuntos
Doença de Alzheimer , Doenças Autoimunes , Humanos , Poliaminas/metabolismo , Doença de Alzheimer/genética , Nucléolo Celular/metabolismo , RNA
20.
Nucleic Acids Res ; 52(8): e41, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38554110

RESUMO

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.


Assuntos
Nucléolo Celular , Núcleo Celular , Sondas de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Sondas de DNA/química , Células HeLa , Chaperonas Moleculares/metabolismo , Avidina/química , Avidina/metabolismo , DNA/metabolismo , Biotina/química
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