Reactivating p53 and Inducing Tumor Apoptosis (RITA) has been reported to increase the p53 activity and to trigger p53-dependent apoptosis in cancer cells with wild-type p53. Tumor suppressor p53 interacts with nucleolar phosphoproteins nucleophosmin (NPM) and nucleolin (NCL), which have crucial role in many cellular processes. Specific NPM mutations associated with acute myeloid leukemia (AML) cause aberrant localization of NPM and p53 in the cytoplasm with possible impact on the p53 function. We tested an effect of RITA on primary cells, and we found significant RITA-induced changes in NPM and NCL phosphorylation associated with apoptosis in cells of AML patients, but not that of healthy donors. Subsequent screening of several AML cell lines revealed heterogeneous response to RITA, and confirmed an association of the specific phosphorylation with apoptosis. While decreased NCL phosphorylation at Threonines T76 and T84 could be attributed to RITA-induced cell cycle arrest, enhanced NPM phosphorylation at Threonine T199 was not accompanied by the cell cycle changes and it correlated with sensitivity to RITA. Simultaneously, inverse changes occurred at Serine S4 of the NPM. These new findings of RITA mechanism of action could establish the NPM pT199/pS4 ratio as a marker for suitability of RITA treatment of AML cells.
Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.
- MeSH
- buněčné jadérko genetika metabolismus MeSH
- DEAD-box RNA-helikasy metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- prekurzory RNA metabolismus MeSH
- ribozomální proteiny * genetika metabolismus MeSH
- ribozomy genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
- MeSH
- axonální transport genetika MeSH
- buněčné jadérko metabolismus ultrastruktura MeSH
- exprese genu MeSH
- fosfoproteiny chemie genetika metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- kineziny genetika metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mutace MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nervus ischiadicus cytologie metabolismus MeSH
- neurony cytologie metabolismus MeSH
- primární buněčná kultura MeSH
- proteinové domény MeSH
- proteiny vázající RNA chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- spinální ganglia cytologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.
- MeSH
- acetylace MeSH
- buněčná inkluze metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- G-kvadruplexy * MeSH
- genetická transkripce * MeSH
- histony metabolismus MeSH
- lidé MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- zastoupení bazí genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Current models of gene expression, which are based on single-molecule localization microscopy, acknowledge protein clustering and the formation of transcriptional condensates as a driving force of gene expression. However, these models largely omit the role of nuclear lipids and amongst them nuclear phosphatidylinositol phosphates (PIPs) in particular. Moreover, the precise distribution of nuclear PIPs in the functional sub-nuclear domains remains elusive. The direct stochastic optical reconstruction microscopy (dSTORM) provides an unprecedented resolution in biological imaging. Therefore, its use for imaging in the densely crowded cell nucleus is desired but also challenging. Here we present a dual-color dSTORM imaging and image analysis of nuclear PI(4,5)P2, PI(3,4)P2 and PI(4)P distribution while preserving the context of nuclear architecture. In the nucleoplasm, PI(4,5)P2 and PI(3,4)P2 co-pattern in close proximity with the subset of RNA polymerase II foci. PI(4,5)P2 is surrounded by fibrillarin in the nucleoli and all three PIPs are dispersed within the matrix formed by the nuclear speckle protein SON. PI(4,5)P2 is the most abundant nuclear PIP, while PI(4)P is a precursor for the biosynthesis of PI(4,5)P2 and PI(3,4)P2. Therefore, our data are relevant for the understanding the roles of nuclear PIPs and provide further evidence for the model in which nuclear PIPs represent a localization signal for the formation of lipo-ribonucleoprotein hubs in the nucleus. The discussed experimental pipeline is applicable for further functional studies on the role of other nuclear PIPs in the regulation of gene expression and beyond.
- MeSH
- buněčné jadérko metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- lidé MeSH
- mikroskopie MeSH
- nádorové buněčné linie MeSH
- RNA-polymerasa II metabolismus MeSH
- vedlejší histokompatibilní antigeny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P's interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P's interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein-lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.
- MeSH
- buněčné jadérko metabolismus ultrastruktura MeSH
- buněčné jádro metabolismus ultrastruktura MeSH
- buněčný cyklus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- jaderný obal metabolismus ultrastruktura MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteom metabolismus MeSH
- shluková analýza MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In nearly all somatic cells, the ribosome biosynthesis is a key activity. The same is true also for mammalian oocytes and early embryos. This activity is intimately linked to the most prominent nuclear organelles - the nucleoli. Interestingly, during a short period around fertilization, the nucleoli in oocytes and embryos transform into ribosome-biosynthesis-inactive structures termed nucleolus-like or nucleolus precursor bodies (NPBs). For decades, researchers considered these structures to be passive repositories of nucleolar proteins used by the developing embryo to rebuild fully functional, ribosome-synthesis competent nucleoli when required. Recent evidence, however, indicates that while these structures are unquestionably essential for development, the material is largely dispensable for the formation of active embryonic nucleoli. In this mini-review, we will describe some unique features of oocytes and embryos with respect to ribosome biogenesis and the changes in the structure of oocyte and embryonic nucleoli that reflect this. We will also describe some of the different approaches that can be used to study nucleoli and NPBs in embryos and discuss the different results that might be expected. Finally, we ask whether the main function of nucleolar precursor bodies might lie in the genome organization and remodelling and what the involved components might be.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- centromera metabolismus MeSH
- embryo savčí cytologie MeSH
- embryonální vývoj genetika MeSH
- fertilizace * MeSH
- histony metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- lidé MeSH
- molekulární chaperony metabolismus MeSH
- oocyty metabolismus MeSH
- organely metabolismus MeSH
- ribozomy metabolismus MeSH
- RNA-polymerasa I metabolismus MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Nucleoli are the site of ribosomal RNA production and subunit assembly. In contrast to active nucleoli in somatic cells, where three basic sub-compartments can be observed, mammalian oocytes and early embryos contain atypical nucleoli termed "nucleolus-like bodies" or "nucleolus precursor bodies", respectively. Unlike their somatic counterparts, these structures are composed of dense homogenous fibrillar material and exhibit no polymerase activity. Irrespective of these unusual properties, they have been shown to be absolutely essential for embryonic development, as their microsurgical removal results in developmental arrest. Historically, nucleolus-like and nucleolus precursor bodies have been perceived as passive storage sites of nucleolar material, which is gradually utilized by embryos to construct fully functional nucleoli once they have activated their genome and have started to produce ribosomes. For decades, researchers have been trying to elucidate the composition of these organelles and provide the evidence for their repository role. However, only recently has it become clear that the function of these atypical nucleoli is altogether different, and rather than being involved in ribosome biogenesis, they participate in parental chromatin remodeling, and strikingly, the artificial introduction of a single NPB component is sufficient to rescue the developmental arrest elicited by the NPB removal. In this review, we will describe and summarize the experiments that led to the change in our understanding of these unique structures.
- MeSH
- buněčné jadérko genetika metabolismus MeSH
- chromatin genetika metabolismus MeSH
- embryonální vývoj genetika MeSH
- lidé MeSH
- restrukturace chromatinu * MeSH
- ribozomy genetika metabolismus MeSH
- zárodečné buňky metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage.
- MeSH
- buněčné jadérko metabolismus MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- fosforylace MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- optické zobrazování MeSH
- poškození DNA MeSH
- rezonanční přenos fluorescenční energie MeSH
- serin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.
- MeSH
- buněčné jadérko metabolismus MeSH
- doxorubicin MeSH
- fyziologický stres MeSH
- kultivované buňky MeSH
- lidé MeSH
- poškození DNA * MeSH
- protein promyelocytické leukemie metabolismus MeSH
- stárnutí buněk * MeSH
- zobrazování trojrozměrné MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
