Topological stress triggers persistent DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment.

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

NAT10 resolves harmful nucleolar R-loops depending on its helicase domain and acetylation of DDX21.

BACKGROUND: Aberrant accumulation of R-loops leads to DNA damage, genome instability and even cell death. Therefore, the timely removal of harmful R-loops is essential for the maintenance of genome integrity. Nucleolar R-loops occupy up to 50% of cellular R-loops due to the frequent activation of Pol I transcription. However, the mechanisms involved in the nucleolar R-loop resolution remain elusive. The nucleolar acetyltransferase NAT10 harbors a putative RecD helicase domain (RHD), however, if NAT10 acts in the R-loop resolution is still unknown. METHODS: NAT10 knockdown cell lines were constructed using CRISPR/Cas9 technology and short hairpin RNA targeting NAT10 mRNA, respectively. The level of R-loops was detected by immunofluorescent staining combined with RNase H treatment. The helicase activity of NAT10 or DDX21 was determined by in vitro helicase experiment. The interaction between NAT10 and DDX21 was verified by co-immunoprecipitation, immunofluorescent staining and GST pull-down experiments. Acetylation sites of DDX21 by NAT10 were analyzed by mass spectrometry. NAT10 knockdown-induced DNA damage was evaluated by immunofluorescent staining and Western blot detecting γH2AX. RESULTS: Depletion of NAT10 led to the accumulation of nucleolar R-loops. NAT10 resolves R-loops through an RHD in vitro and in cells. However, Flag-NAT10 ∆RHD mutant still partially reduced R-loop levels in the NAT10-depleted cells, suggesting that NAT10 might resolve R-loops through additional pathways. Further, the acetyltransferase activity of NAT10 is required for the nucleolar R-loop resolution. NAT10 acetylates DDX21 at K236 and K573 to enhance the helicase activity of DDX21 to unwind nucleolar R-loops. The helicase activity of DDX21 significantly decreased by Flag-DDX21 2KR and increased by Flag-DDX21 2KQ in cells and in vitro. Consequently, NAT10 depletion-induced nucleolar R-loop accumulation led to DNA damage, which was rescued by co-expression of Flag-DDX21 2KQ and Flag-NAT10 G641E, demonstrating that NAT10 resolves nucleolar R-loops through bipartite pathways. CONCLUSION: We demonstrate that NAT10 is a novel R-loop resolvase and it resolves nucleolar R-loops depending on its helicase activity and acetylation of DDX21. The cooperation of NAT10 and DDX21 provides comprehensive insights into the nucleolar R-loop resolution for maintaining genome stability.

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments.

Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.

The Nucleolus and Its Interactions with Viral Proteins Required for Successful Infection.

Nuclear bodies are structures in eukaryotic cells that lack a plasma membrane and are considered protein condensates, DNA, or RNA molecules. Known nuclear bodies include the nucleolus, Cajal bodies, and promyelocytic leukemia nuclear bodies. These bodies are involved in the concentration, exclusion, sequestration, assembly, modification, and recycling of specific components involved in the regulation of ribosome biogenesis, RNA transcription, and RNA processing. Additionally, nuclear bodies have been shown to participate in cellular processes such as the regulation of transcription of the cell cycle, mitosis, apoptosis, and the cellular stress response. The dynamics and functions of these bodies depend on the state of the cell. It is now known that both DNA and RNA viruses can direct their proteins to nuclear bodies, causing alterations in their composition, dynamics, and functions. Although many of these mechanisms are still under investigation, it is well known that the interaction between viral and nuclear body proteins is necessary for the success of the viral infection cycle. In this review, we concisely describe the interaction between viral and nuclear body proteins. Furthermore, we focus on the role of the nucleolus in RNA virus infections. Finally, we discuss the possible implications of the interaction of viral proteins on cellular transcription and the formation/degradation of non-coding RNAs.

SURF2 is a MDM2 antagonist in triggering the nucleolar stress response.

Cancer cells rely on high ribosome production to sustain their proliferation rate. Many chemotherapies impede ribosome production which is perceived by cells as "nucleolar stress" (NS), triggering p53-dependent and independent pathways leading to cell cycle arrest and/or apoptosis. The 5S ribonucleoprotein (RNP) particle, a sub-ribosomal particle, is instrumental to NS response. Upon ribosome assembly defects, the 5S RNP accumulates as free form. This free form is able to sequester and inhibit MDM2, thus promoting p53 stabilization. To investigate how cancer cells can resist to NS, here we purify free 5S RNP and uncover an interaction partner, SURF2. Functional characterization of SURF2 shows that its depletion increases cellular sensitivity to NS, while its overexpression promotes their resistance to it. Consistently, SURF2 is overexpressed in many cancers and its expression level is an independent marker of prognosis for adrenocortical cancer. Our data demonstrate that SURF2 buffers free 5S RNP particles, and can modulate their activity, paving the way for the research of new molecules that can finely tune the response to nucleolar stress in the framework of cancer therapies.

Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.

SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

Protein UFMylation regulates early events during ribosomal DNA-damage response.

The highly repetitive and transcriptionally active ribosomal DNA (rDNA) genes are exceedingly susceptible to genotoxic stress. Induction of DNA double-strand breaks (DSBs) in rDNA repeats is associated with ataxia-telangiectasia-mutated (ATM)-dependent rDNA silencing and nucleolar reorganization where rDNA is segregated into nucleolar caps. However, the regulatory events underlying this response remain elusive. Here, we identify protein UFMylation as essential for rDNA-damage response in human cells. We further show the only ubiquitin-fold modifier 1 (UFM1)-E3 ligase UFL1 and its binding partner DDRGK1 localize to nucleolar caps upon rDNA damage and that UFL1 loss impairs ATM activation and rDNA transcriptional silencing, leading to reduced rDNA segregation. Moreover, analysis of nuclear and nucleolar UFMylation targets in response to DSB induction further identifies key DNA-repair factors including ATM, in addition to chromatin and actin network regulators. Taken together, our data provide evidence of an essential role for UFMylation in orchestrating rDNA DSB repair.

To the ring-shaped nucleolus seen by microscopy using human lymphocytes of blood donors and chronic lymphocytic leukemia patients.

The present study was undertaken to provide more information on the peripheral RNA containing ring of ringshaped nucleoli (RSNo). Human lymphocytes of blood donors and patients suffering from B chronic lymphocytic leukemia mostly characterized by RSNo represented very convenient cell models for such study. According to the light microscopy the peripheral RNA ring possessed several highly condensed foci. Such regions represented accumulated dense RNA fibrillar components (DFCs) seen by the electron microscopy. In contrary, the incidence of dense granular RNA-containing components (GCs) in surrounding portions of the RNA ring was small. Thus, the structural and morphological organization of the peripheral RNA ring of RSNo apparently reflects sites of micro-segregated foci of DFCs and a small incidence of GCs. That structural organization of the peripheral RNA ring of RSNo appeared to be a prerequisite for further regressive nucleolar changes resulting in the development of micronucleoli in terminal lymphocytes.

Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated Tyramide Signal Amplification (TSA)-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in human embryonic stem cells (hESCs). Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation.

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.

Nucleolar stress induces nucleolar stress body formation via the NOSR-1/NUMR-1 axis in Caenorhabditis elegans.

Environmental stimuli not only alter gene expression profiles but also induce structural changes in cells. How distinct nuclear bodies respond to cellular stress is poorly understood. Here, we identify a subnuclear organelle named the nucleolar stress body (NoSB), the formation of which is induced by the inhibition of rRNA transcription or inactivation of rRNA processing and maturation in C. elegans. NoSB does not colocalize with other previously described subnuclear organelles. We conduct forward genetic screening and identify a bZIP transcription factor, named nucleolar stress response-1 (NOSR-1), that is required for NoSB formation. The inhibition of rRNA transcription or inactivation of rRNA processing and maturation increases nosr-1 expression. By using transcriptome analysis of wild-type animals subjected to different nucleolar stress conditions and nosr-1 mutants, we identify that the SR-like protein NUMR-1 (nuclear localized metal responsive) is the target of NOSR-1. Interestingly, NUMR-1 is a component of NoSB and itself per se is required for the formation of NoSB. We conclude that the NOSR-1/NUMR-1 axis likely responds to nucleolar stress and mediates downstream stress-responsive transcription programs and subnuclear morphology alterations in C. elegans.

Single-cell laser emitting cytometry for label-free nucleolus fingerprinting.

The nucleolus, a recognized biomolecular condensate, serves as the hub for ribosome biogenesis within the cell nucleus. Its quantity and morphology are discernible indicators of cellular functional states. However, precise identification and quantification of nucleoli remain challenging without specific labeling, particularly for suspended cells, tissue-level analysis and high-throughput applications. Here we introduce a single-cell laser emitting cytometry (SLEC) for label-free nucleolus differentiation through light-matter interactions within a Fabry-Perot resonator. The separated gain medium enhances the threshold difference by 36-fold between nucleolus and its surroundings, enabling selective laser emissions at nucleolar area while maintaining lower-order mode. The laser emission image provides insights into structural inhomogeneity, temporal fluid-like dynamics, and pathological application. Lasing spectral fingerprint depicts the quantity and size of nucleoli within a single cell, showcasing the label-free flow cytometry for nucleolus. This approach holds promise for nucleolus-guided cell screening and drug evaluation, advancing the study of diseases such as cancer and neurodegenerative disorders.

Developmental analysis of Spalt function in the Drosophila prothoracic gland.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

Putting It All Together: The Roles of Ribosomal Proteins in Nucleolar Stages of 60S Ribosomal Assembly in the Yeast Saccharomyces cerevisiae.

Here we review the functions of ribosomal proteins (RPs) in the nucleolar stages of large ribosomal subunit assembly in the yeast Saccharomyces cerevisiae. We summarize the effects of depleting RPs on pre-rRNA processing and turnover, on the assembly of other RPs, and on the entry and exit of assembly factors (AFs). These results are interpreted in light of recent near-atomic-resolution cryo-EM structures of multiple assembly intermediates. Results are discussed with respect to each neighborhood of RPs and rRNA. We identify several key mechanisms related to RP behavior. Neighborhoods of RPs can assemble in one or more than one step. Entry of RPs can be triggered by molecular switches, in which an AF is replaced by an RP binding to the same site. To drive assembly forward, rRNA structure can be stabilized by RPs, including clamping rRNA structures or forming bridges between rRNA domains.

Nucleophosmin: A Nucleolar Phosphoprotein Orchestrating Cellular Stress Responses.

Nucleophosmin (NPM1) is a key nucleolar protein released from the nucleolus in response to stress stimuli. NPM1 functions as a stress regulator with nucleic acid and protein chaperone activities, rapidly shuttling between the nucleus and cytoplasm. NPM1 is ubiquitously expressed in tissues and can be found in the nucleolus, nucleoplasm, cytoplasm, and extracellular environment. It plays a central role in various biological processes such as ribosome biogenesis, cell cycle regulation, cell proliferation, DNA damage repair, and apoptosis. In addition, it is highly expressed in cancer cells and solid tumors, and its mutation is a major cause of acute myeloid leukemia (AML). This review focuses on NPM1's structural features, functional diversity, subcellular distribution, and role in stress modulation.

SNORD88B-mediated WRN nucleolar trafficking drives self-renewal in liver cancer initiating cells and hepatocarcinogenesis.

Whether small nucleolar RNAs (snoRNAs) are involved in the regulation of liver cancer stem cells (CSCs) self-renewal and serve as therapeutic targets remains largely unclear. Here we show that a functional snoRNA (SNORD88B) is robustly expressed in Hepatocellular carcinoma (HCC) tumors and liver CSCs. SNORD88B deficiency abolishes the self-renewal of liver CSCs and hepatocarcinogenesis. Mechanistically, SNORD88B anchors WRN in the nucleolus, promoting XRCC5 interacts with STK4 promoter to suppress its transcription, leading to inactivation of Hippo signaling. Moreover, low expression of STK4 and high expression of XRCC5 are positively correlated with HCC poor prognosis. Additionally, snord88b knockout suppresses mouse liver tumorigenesis. Notably, co-administration of SNORD88B antisense oligonucleotides (ASOs) with MST1 agonist adapalene (ADA) exert synergistic antitumor effects and increase overall murine survival. Our findings delineate that SNORD88B drives self-renewal of liver CSCs and accelerates HCC tumorigenesis via non-canonical mechanism, providing potential targets for liver cancer therapy by eliminating liver CSCs.

METTL3/METTL14 maintain human nucleoli integrity by mediating SUV39H1/H2 degradation.

Nucleoli are fundamentally essential sites for ribosome biogenesis in cells and formed by liquid-liquid phase separation (LLPS) for a multilayer condensate structure. How the nucleoli integrity is maintained remains poorly understood. Here, we reveal that METTL3/METTL14, the typical methyltransferase complex catalyzing N6-methyladnosine (m6A) on mRNAs maintain nucleoli integrity in human embryonic stem cells (hESCs). METTL3/METTL14 deficiency impairs nucleoli and leads to the complete loss of self-renewal in hESCs. We further show that SUV39H1/H2 protein, the methyltransferases catalyzing H3K9me3 were dramatically elevated in METTL3/METTL14 deficient cells, which causes an accumulation and infiltration of H3K9me3 across the whole nucleolus and impairs the LLPS. Mechanistically, METTL3/METTL14 complex serves as an essential adapter for CRL4 E3 ubiquitin ligase targeting SUV39H1/H2 for polyubiquitination and proteasomal degradation and therefore prevents H3K9me3 accumulation in nucleoli. Together, these findings uncover a previously unknown role of METTL3/METTL14 to maintain nucleoli integrity by facilitating SUV39H1/H2 degradation in human cells.

Maize Dek51 encodes a DEAD-box RNA helicase essential for pre-rRNA processing and seed development.

Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3' end processing of 18S and 5.8S pre-rRNA in maize (Zea mays L.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3' end processing of pre-rRNA.

Reduced dynamicity and increased high-order protein assemblies in dense fibrillar component of the nucleolus under cellular senescence.

Cellular senescence, which is triggered by various stressors, manifests as irreversible cell cycle arrest, resulting in the disruption of multiple nuclear condensates. One of the affected structures is the nucleolus, whose tripartite layout, separated into distinct liquid phases, allows for the stepwise progression of ribosome biogenesis. The dynamic properties of dense fibrillar components, a sub-nucleolar phase, are crucial for mediating pre-rRNA processing. However, the mechanistic link between the material properties of dense fibrillar components and cellular senescence remains unclear. We established a significant association between cellular senescence and alterations in nucleolar materiality and characteristics, including the number, size, and sphericity of individual subphases of the nucleolus. Senescent cells exhibit reduced fibrillarin dynamics, aberrant accumulation of high-order protein assemblies, such as oligomers and fibrils, and increased dense fibrillar component density. Intriguingly, the addition of RNA-interacting entities mirrored the diminished diffusion of fibrillarin in the nucleolus during cellular senescence. Thus, our findings contribute to a broader understanding of the intricate changes in the materiality of the nucleolus associated with cellular senescence and shed light on nucleolar dynamics in the context of aging and cellular stress.