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1.
Int J Mol Sci ; 25(12)2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38928039

ABSTRACT

Different developmental genes shape frequent dynamic inter-chromosomal contacts with rDNA units in human and Drosophila cells. In the course of differentiation, changes in these contacts occur, coupled with changes in the expression of hundreds of rDNA-contacting genes. The data suggest a possible role of nucleoli in the global regulation of gene expression. However, the mechanism behind the specificity of these inter-chromosomal contacts, which are rebuilt in every cell cycle, is not yet known. Here, we describe the strong association of rDNA-contacting genes with numerous long intergenic non-coding RNAs (lincRNAs) in HEK293T cells and in initial and differentiated K562 cells. We observed that up to 600 different lincRNAs were preferentially co-expressed with multiple overlapping sets of rDNA-contacting developmental genes, and there was a strong correlation between the genomic positions of rDNA-contacting genes and lincRNA mappings. These two findings suggest that lincRNAs might guide the corresponding developmental genes toward rDNA clusters. We conclude that the inter-chromosomal interactions of rDNA-contacting genes with nucleoli might be guided by lincRNAs, which might physically link particular genomic regions with rDNA clusters.


Subject(s)
Cell Nucleolus , DNA, Ribosomal , RNA, Long Noncoding , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , HEK293 Cells , K562 Cells
2.
Nat Cell Biol ; 26(6): 946-961, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38745030

ABSTRACT

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.


Subject(s)
Chromosomal Proteins, Non-Histone , Leukemia, Myeloid, Acute , RNA Precursors , RNA Processing, Post-Transcriptional , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , RNA Precursors/metabolism , RNA Precursors/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Animals , Cell Line, Tumor , Protein Biosynthesis , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , Mice , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Phase Separation
3.
J Cell Biol ; 223(7)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38717338

ABSTRACT

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.


Subject(s)
DNA Helicases , Multifunctional Enzymes , RNA Helicases , RNA, Untranslated , Humans , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , DNA Damage , DNA Helicases/metabolism , DNA Helicases/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , Protein Aggregates , Proteostasis , R-Loop Structures/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , RNA, Untranslated/genetics , RNA, Untranslated/metabolism
4.
J Ovarian Res ; 17(1): 99, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38730385

ABSTRACT

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.


Subject(s)
Chromatin , Oocytes , Transcriptome , Humans , Oocytes/metabolism , Chromatin/metabolism , Chromatin/genetics , Female , Gene Expression Profiling , Cell Nucleolus/metabolism , Cell Nucleolus/genetics
5.
Nucleic Acids Res ; 52(11): 6360-6375, 2024 Jun 24.
Article in English | MEDLINE | ID: mdl-38682589

ABSTRACT

Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell suppression remain unclear. Here, we observed that a low dose of DNA-PK-i and PARP inhibitor (PARP-i) synthetically suppresses BRCA-deficient tumor cells without inducing DNA double-strand breaks (DSBs). Instead, we found that a fraction of DNA-PK localized inside of nucleoli, where we did not observe obvious DSBs. Moreover, the Ku proteins recognize pre-rRNA that facilitates DNA-PKcs autophosphorylation independent of DNA damage. Ribosomal proteins are also phosphorylated by DNA-PK, which regulates pre-rRNA biogenesis. In addition, DNA-PK-i acts together with PARP-i to suppress pre-rRNA biogenesis and tumor cell growth. Collectively, our studies reveal a DNA damage repair-independent role of DNA-PK-i in tumor suppression.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , DNA-Activated Protein Kinase , Ku Autoantigen , RNA Precursors , DNA-Activated Protein Kinase/metabolism , DNA-Activated Protein Kinase/genetics , Humans , RNA Precursors/metabolism , RNA Precursors/genetics , Cell Line, Tumor , Ku Autoantigen/metabolism , Ku Autoantigen/genetics , Phosphorylation , Cell Nucleolus/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/drug effects , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , RNA, Ribosomal/metabolism , RNA, Ribosomal/genetics , Animals , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism
6.
Mol Cell ; 84(8): 1400-1402, 2024 Apr 18.
Article in English | MEDLINE | ID: mdl-38640892

ABSTRACT

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.


Subject(s)
Cell Nucleolus , Ribosomal Proteins , Mice , Animals , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , RNA, Ribosomal/metabolism
7.
Mol Cell ; 84(8): 1527-1540.e7, 2024 Apr 18.
Article in English | MEDLINE | ID: mdl-38521064

ABSTRACT

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.


Subject(s)
Neoplasms , Ribosomes , Mice , Animals , Ribosomes/metabolism , Aging/genetics , Peptides/metabolism , Sirolimus/pharmacology , Neoplasms/metabolism , Cell Nucleolus/genetics , Mammals
8.
Elife ; 122024 Mar 26.
Article in English | MEDLINE | ID: mdl-38530350

ABSTRACT

Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.


Subject(s)
Cell Nucleus , Histones , Humans , Histones/genetics , Cell Nucleolus/genetics , Chromatin , Image Processing, Computer-Assisted
9.
J Biol Chem ; 300(3): 105773, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38382671

ABSTRACT

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.


Subject(s)
Cell Nucleolus , DNA-Binding Proteins , Nucleophosmin , Transcription Factors , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Nuclear Proteins/metabolism , Protein Binding , RNA, Ribosomal/metabolism , Humans , Transcription Factors/metabolism , DNA-Binding Proteins/metabolism
10.
Elife ; 132024 Jan 19.
Article in English | MEDLINE | ID: mdl-38240312

ABSTRACT

Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.


Subject(s)
Nucleolus Organizer Region , RNA Precursors , Humans , Animals , Nucleolus Organizer Region/genetics , Nucleolus Organizer Region/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Chromosomes, Human/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Mammals/genetics
11.
FEBS Lett ; 598(2): 187-198, 2024 01.
Article in English | MEDLINE | ID: mdl-38058218

ABSTRACT

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM-like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional repression. These results demonstrate that this NPM-like protein relies on IDRs only for some of its chromatin-related functions.


Subject(s)
Histone Chaperones , Lysine , Histone Chaperones/genetics , Histone Chaperones/metabolism , Nucleoplasmins/metabolism , Lysine/metabolism , Chromatin/genetics , Chromatin/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
12.
FEBS Lett ; 598(3): 283-301, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37994551

ABSTRACT

Reprogramming organelle size has been proposed as a potential therapeutic approach. However, there have been few reports of nucleolar size reprogramming. We addressed this question in Saccharomyces cerevisiae by studying mutants having opposite effects on the nucleolar size. Mutations in genes involved in nuclear functions (KAR3, CIN8, and PRP45) led to enlarged nuclei/nucleoli, whereas mutations in secretory pathway family genes, namely the Rab-GTPases YPT6 and YPT32, reduced nucleolar size. When combined with mutations leading to enlarged nuclei/nucleoli, the YPT6 or YPT32 mutants can effectively reprogram the nuclear/nucleolar size almost back to normal. Our results further indicate that null mutation of YPT6 causes secretory stress that indirectly influences nuclear localization of Maf1, the negative regulator of RNA Polymerase III, which might reduce the nucleolar size by inhibiting nucleolar transcript enrichment.


Subject(s)
Monomeric GTP-Binding Proteins , Saccharomyces cerevisiae Proteins , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Mutation , Biological Transport , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Kinesins/metabolism , Monomeric GTP-Binding Proteins/metabolism
13.
Commun Biol ; 6(1): 1129, 2023 11 07.
Article in English | MEDLINE | ID: mdl-37935838

ABSTRACT

Liquid-liquid phase separation (LLPS) has been thought to be the biophysical principle governing the assembly of the multiphase structures of nucleoli, the site of ribosomal biogenesis. Condensates assembled through LLPS increase their sizes to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their sizes do not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the FC growth, we here construct a minimal theoretical model by taking into account nascent pre-rRNAs tethered to FC surfaces by RNA polymerase I. The prediction of this theory was supported by our experiments that quantitatively measure the dependence of the size of FCs on the transcription level. This work sheds light on the role of nascent RNAs in controlling the size of nuclear bodies.


Subject(s)
Pulmonary Surfactants , RNA, Ribosomal , RNA, Ribosomal/genetics , RNA, Ribosomal/analysis , Surface-Active Agents , Cell Nucleolus/chemistry , Cell Nucleolus/genetics , RNA/genetics , RNA/analysis , RNA Precursors/genetics , RNA Precursors/analysis
14.
Sci Adv ; 9(44): eadj4509, 2023 11 03.
Article in English | MEDLINE | ID: mdl-37910609

ABSTRACT

Arabidopsis thaliana has two ribosomal RNA (rRNA) gene loci, nucleolus organizer regions NOR2 and NOR4, whose complete sequences are missing in current genome assemblies. Ultralong DNA sequences assembled using an unconventional approach yielded ~5.5- and 3.9-Mbp sequences for NOR2 and NOR4 in the reference strain, Col-0. The distinct rRNA gene subtype compositions of the NORs enabled the positional mapping of their active and inactive regions, using RNA sequencing to identify subtype-specific transcripts and DNA sequencing to identify subtypes associated with flow-sorted nucleoli. Comparisons of wild-type and silencing-defective plants revealed that most rRNA gene activity occurs in the central region of NOR4, whereas most, but not all, genes of NOR2 are epigenetically silenced. Intervals of low CG and CHG methylation overlap regions where gene activity and gene subtype homogenization are high. Collectively, the data reveal the genetic and epigenetic landscapes underlying nucleolar dominance (differential NOR activity) and implicate transcription as a driver of rRNA gene concerted evolution.


Subject(s)
Arabidopsis , Nucleolus Organizer Region , Nucleolus Organizer Region/genetics , Arabidopsis/genetics , RNA, Ribosomal/genetics , Cell Nucleolus/genetics , Epigenesis, Genetic
15.
Mol Cell ; 83(23): 4413-4423.e10, 2023 Dec 07.
Article in English | MEDLINE | ID: mdl-37979585

ABSTRACT

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.


Subject(s)
DEAD-box RNA Helicases , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/chemistry , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Organelles/metabolism , Hydrogen-Ion Concentration
16.
Nucleus ; 14(1): 2274655, 2023 12.
Article in English | MEDLINE | ID: mdl-37906621

ABSTRACT

The nucleolus, the largest subcompartment of the nucleus, stands out from the nucleoplasm due to its exceptionally high local RNA and low DNA concentrations. Within this central hub of nuclear RNA metabolism, ribosome biogenesis is the most prominent ribonucleoprotein (RNP) biogenesis process, critically determining the structure and function of the nucleolus. However, recent studies have shed light on other roles of the nucleolus, exploring the interplay with various noncoding RNAs that are not directly involved in ribosome synthesis. This review focuses on this intriguing topic and summarizes the techniques to study and the latest findings on nucleolar long noncoding RNAs (lncRNAs) as well as microRNAs (miRNAs) in the context of nucleolus biology beyond ribosome biogenesis. We particularly focus on the multifaceted roles of the nucleolus and noncoding RNAs in physiology and tumor biology.


Subject(s)
Neoplasms , RNA, Long Noncoding , Humans , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Biology
17.
Mol Cell ; 83(17): 3095-3107.e9, 2023 09 07.
Article in English | MEDLINE | ID: mdl-37683610

ABSTRACT

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 (∼1 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.


Subject(s)
RNA, Ribosomal , RNA , RNA/genetics , RNA, Ribosomal/genetics , Biomolecular Condensates , Cell Nucleolus/genetics , Nuclear Proteins/genetics
18.
PLoS Genet ; 19(8): e1010854, 2023 08.
Article in English | MEDLINE | ID: mdl-37639467

ABSTRACT

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.


Subject(s)
Cell Nucleolus , Cell Nucleus Division , Cell Nucleolus/genetics , Cell Cycle , Cell Proliferation , RNA Polymerase I/genetics , RNA, Ribosomal/genetics
19.
Parasitol Res ; 122(9): 1961-1971, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37400534

ABSTRACT

Giardia duodenalis is a protozoan intestinal parasite that causes a significant number of infections worldwide each year, particularly in low-income and developing countries. Despite the availability of treatments for this parasitic infection, treatment failures are alarmingly common. As a result, new therapeutic strategies are urgently needed to effectively combat this disease. On the other hand, within the eukaryotic nucleus, the nucleolus stands out as the most prominent structure. It plays a crucial role in coordinating ribosome biogenesis and is involved in vital processes such as maintaining genome stability, regulating cell cycle progression, controlling cell senescence, and responding to stress. Given its significance, the nucleolus presents itself as a valuable target for selectively inducing cell death in undesirable cells, making it a potential avenue for anti-Giardia treatments. Despite its potential importance, the Giardia nucleolus remains poorly studied and often overlooked. In light of this, the objective of this study is to provide a detailed molecular description of the structure and function of the Giardia nucleolus, with a primary focus on its involvement in ribosomal biogenesis. Likewise, it discusses the targeting of the Giardia nucleolus as a therapeutic strategy, its feasibility, and the challenges involved.


Subject(s)
Cell Nucleolus , Giardia , Ribosomes , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Giardia/cytology , Giardia/genetics , RNA, Ribosomal/genetics , DNA, Ribosomal/genetics , DNA, Protozoan/genetics , RNA, Protozoan/genetics , Transcription, Genetic , Gene Expression Regulation , RNA Processing, Post-Transcriptional/genetics , Ribosomes/genetics , Ribosomes/metabolism , Giardiasis/drug therapy , Antiparasitic Agents/therapeutic use , Drug Development/trends
20.
Genetics ; 225(1)2023 08 31.
Article in English | MEDLINE | ID: mdl-37433110

ABSTRACT

Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in Caenorhabditis elegans that is marked by abnormal globular morphology of the nucleolus and fewer germ cells. We identified DDB1 Cullin4 associated factor (DCAF)-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, the inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of the germ line-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor periodic trptophan protein 1 (PWP1). We observed that the inactivation of Caenorhabditis elegansDCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.


Subject(s)
Caenorhabditis elegans , Cullin Proteins , Male , Animals , Mice , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cullin Proteins/genetics , Cullin Proteins/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Ubiquitin/metabolism , Semen/metabolism , Germ Cells/metabolism , Transcription Factors/genetics
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