Nucleolar Function Research Articles

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer, glioblastoma (GBM). This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein, Nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in GBM reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of GBM cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for aberrant nucleolar function and increased anabolic processes in GBM, which constitutes a primary event in gliomagenesis.

PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H2O2, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H2O2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.

Emerging Role of the Nucleolar Stress Response in Autophagy.

Autophagy represents a conserved self-digestion program, which allows regulated degradation of cellular material. Autophagy is activated by cellular stress, serum starvation and nutrient deprivation. Several autophagic pathways have been uncovered, which either non-selectively or selectively target the cellular cargo for lysosomal degradation. Autophagy engages the coordinated action of various key regulators involved in the steps of autophagosome formation, cargo targeting and lysosomal fusion. While non-selective (macro)autophagy is required for removal of bulk material or recycling of nutrients, selective autophagy mediates specific targeting of damaged organelles or protein aggregates. By proper action of the autophagic machinery, cellular homeostasis is maintained. In contrast, failure of this fundamental process is accompanied by severe pathophysiological conditions. Hallmarks of neuropathological disorders are for instance accumulated, mis-folded protein aggregates and damaged mitochondria. The nucleolus has been recognized as central hub in the cellular stress response. It represents a sub-nuclear organelle essential for ribosome biogenesis and also functions as stress sensor by mediating cell cycle arrest or apoptosis. Thus, proper nucleolar function is mandatory for cell growth and survival. Here, I highlight the emerging role of nucleolar factors in the regulation of autophagy. Moreover, I discuss the nucleolar stress response as a novel signaling pathway in the context of autophagy, health and disease.

Phase-to-Phase With Nucleoli - Stress Responses, Protein Aggregation and Novel Roles of RNA.

Protein- and RNA-containing foci and aggregates are a hallmark of many age- and mutation-related neurodegenerative diseases. This article focuses on the role the nucleolus has as a hub in macromolecule regulation in the mammalian nucleus. The nucleolus has a well-established role in ribosome biogenesis and functions in several types of cellular stress responses. In addition to known reactions to DNA damaging and transcription inhibiting stresses, there is an emerging role of the nucleolus especially in responses to proteotoxic stress such as heat shock and inhibition of proteasome function. The nucleolus serves as an active regulatory site for detention of extranucleolar proteins. This takes place in nucleolar cavities and manifests in protein and RNA collections referred to as intranucleolar bodies (INBs), nucleolar aggresomes or amyloid bodies (A-bodies), depending on stress type, severity of accumulation, and material propensities of the macromolecular collections. These indicate a relevance of nucleolar function and regulation in neurodegeneration-related cellular events, but also provide surprising connections with cancer-related pathways. Yet, the molecular mechanisms governing these processes remain largely undefined. In this article, the nucleolus as the site of protein and RNA accumulation and as a possible protective organelle for nuclear proteins during stress is viewed. In addition, recent evidence of liquid-liquid phase separation (LLPS) and liquid-solid phase transition in the formation of nucleoli and its stress responses, respectively, are discussed, along with the increasingly indicated role and open questions for noncoding RNA species in these events.

Nucleolar protein nucleolin functions in replication stress-induced DNA damage responses.

The nucleolus contains multiple copies of ribosomal (r)DNA, which indicate sites of frequent replication stress and suggest the existence of a mechanism to prevent replication stress–related rDNA instability and the possibility that such a mechanism contributes to the whole genomic stability against replication stress. We have previously reported that nucleolin, a major nucleolar protein, is involved in ionizing radiation–induced DNA damage responses (DDRs) such as ataxia telangiectasia mutated (ATM)-dependent cell cycle checkpoints and homologous recombination (HR) repair. Here, we investigated the role of nucleolin in DDR due to replication stress. The results indicate that following replication stress, nucleolin interacted with the histone γH2AX, proliferating cell nuclear antigen (PCNA), and replication protein A (RPA)32, suggesting that it may be recruited to DNA damage sites on the replication fork. Furthermore, the knockdown of nucleolin by siRNA reduced the activation of ATM and RAD3-related (ATR) kinase and the formation of RAD51 and RPA32 foci after replication stress due to UV or camptothecin exposure, whereas nucleolin overexpression augmented ATR-dependent phosphorylation and RAD51 and RPA accumulation on chromatin. Moreover, these overexpressing cells seemed to increase repair activity and resistance to replication stress. Our results indicate that nucleolin plays an important role in replication stress–induced DDRs such as ATR activation and HR repair. Given that nucleolin overexpression is often observed in many types of cancer cells, our findings suggest that nucleolin is involved in the regulation of resistance to replication stress that may otherwise lead to tumorigenesis and it could be a possible target for chemotherapy and radiotherapy.

Altered Nucleolar Phase Separation by NPM1 in ALS

Amyotrophic lateral sclerosis (ALS) is a lethal and incurable neurodegenerative disorder commonly associated with repeat expansion in the C9orf72 gene; termed C9-ALS. A major pathological feature of this is the accumulation of arginine-rich (R-rich) dipeptide repeat (DPR) polypeptides in neurons. R-rich DPRs interact with low complexity domains in proteins, accumulate in membraneless organelles (MLOs), alter the material properties of MLOs, and induce cell death. The molecular mechanisms underlying pathogenesis, though, remain unknown. R-rich DPRs infiltrate nucleoli, co-localize with nucleophosmin (NPM1), and alter NPM1 phase separation in vitro. NPM1 is crucial to the maintenance of nucleolar liquid-like properties through its ability to phase separate with proteins and nucleic acids. Elucidating the effects of DPR interactions with NPM1 on the liquid-like properties and overall architecture of nucleoli that ultimately lead to nucleolar dysfunction and cell death is critical to understanding C9-ALS. Here we employ poly(PR) as an archetypal R-rich DPR to (1) identify the interactions mediating phase separation with NPM1; (2) elucidate the mechanisms causing the DPR-dependent dissolution of in vitro NPM1/DPR droplets; and (3) confirm hypotheses stimulated by our biophysical results regarding the mechanisms of DPR-mediated nucleolar disruption in cells. Results from multiple, complementary biochemical and biophysical techniques show that (1) NPM1/DPR interactions are mediated by acidic tracts within the intrinsically disorder region of NPM1, (2) R-rich DPRs dissolve NPM1-containing droplets in vitro by sequestering NPM1 into large saturated complexes, and (3) exogenous poly(PR) induces NPM1 release from nucleoli, disrupting nucleolar organization and function. These results support the hypothesis that R-rich DPRs, mediate their toxic effects in part through saturation/sequestration of NPM1, perturbing NPM1 mediated phase separation in nucleoli, disrupting nucleolar function, and inducing cell death.

The Ribosome Biogenesis-Cancer Connection.

Multifaceted relations link ribosome biogenesis to cancer. Ribosome biogenesis takes place in the nucleolus. Clarifying the mechanisms involved in this nucleolar function and its relationship with cell proliferation: (1) allowed the understanding of the reasons for the nucleolar changes in cancer cells and their exploitation in tumor pathology, (2) defined the importance of the inhibition of ribosome biogenesis in cancer chemotherapy and (3) focused the attention on alterations of ribosome biogenesis in the pathogenesis of cancer. This review summarizes the research milestones regarding these relevant relationships between ribosome biogenesis and cancer. The structure and function of the nucleolus will also be briefly described.

Molecular Mechanisms of Neurodegeneration Related to C9orf72 Hexanucleotide Repeat Expansion.

Two clinically distinct diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), have recently been classified as two extremes of the FTD/ALS spectrum. The neuropathological correlate of FTD is frontotemporal lobar degeneration (FTLD), characterized by tau-, TDP-43-, and FUS-immunoreactive neuronal inclusions. An earlier discovery that a hexanucleotide repeat expansion mutation in chromosome 9 open reading frame 72 (C9orf72) gene causes ALS and FTD established a special subtype of ALS and FTLD with TDP-43 pathology (C9FTD/ALS). Normal individuals carry 2–10 hexanucleotide GGGGCC repeats in the C9orf72 gene, while more than a few hundred repeats represent a risk for ALS and FTD. The proposed molecular mechanisms by which C9orf72 repeat expansions induce neurodegenerative changes are C9orf72 loss-of-function through haploinsufficiency, RNA toxic gain-of-function, and gain-of-function through the accumulation of toxic dipeptide repeat proteins. However, many more cellular processes are affected by pathological processes in C9FTD/ALS, including nucleocytoplasmic transport, RNA processing, normal function of nucleolus, formation of membraneless organelles, translation, ubiquitin proteasome system, Notch signalling pathway, granule transport, and normal function of TAR DNA-binding protein 43 (TDP-43). Although the exact molecular mechanisms through which C9orf72 repeat expansions account for neurodegeneration have not been elucidated, some potential therapeutics, such as antisense oligonucleotides targeting hexanucleotide GGGGCC repeats in mRNA, were successful in preclinical trials and are awaiting phase 1 clinical trials. In this review, we critically discuss each proposed mechanism and provide insight into the most recent studies aiming to elucidate the molecular underpinnings of C9FTD/ALS.

Control of nucleolar stress and translational reprogramming by lncRNAs.

Under adverse environmental conditions, cells activate stress re-sponses that favour adaptation or, in case of irreversible damage, induce cell death. Multiple stress response pathways converge to downregulate ribo-some biogenesis and translation since these are the most energy consuming processes in the cell. This adaptive response allows preserving genomic stabil-ity and saving energy for the recovery. It follows that the nucleolus is a major sensor and integrator of stress responses that are then transmitted to the translation machinery through an intricate series of conserved events. Long non-coding RNAs (lncRNAs) are emerging as important regulators of stress-induced cascades, for their ability to mediate post-transcriptional responses. Consistently, many of them are specifically expressed under stress conditions and a few have been already functionally linked to these processes, thus fur-ther supporting a role in stress management. In this review we survey differ-ent archetypes of lncRNAs specifically implicated in the regulation of nucleo-lar functions and translation reprogramming during stress responses.

Inhibition of nucleolar stress response by Sirt1: A potential mechanism of acetylation-independent regulation of p53 accumulation.

The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein–protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress‐induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1‐mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.

Tumor suppressor PNRC1 blocks rRNA maturation by recruiting the decapping complex to the nucleolus.

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

Nucleolar Stress: hallmarks, sensing mechanism and diseases.

The nucleolus is a prominent subnuclear compartment, where ribosome biosynthesis takes place. Recently, the nucleolus has gained attention for its novel role in the regulation of cellular stress. Nucleolar stress is emerging as a new concept, which is characterized by diverse cellular insult-induced abnormalities in nucleolar structure and function, ultimately leading to activation of p53 or other stress signaling pathways and alterations in cell behavior. Despite a number of comprehensive reviews on this concept, straightforward and clear-cut way criteria for a nucleolar stress state, regarding the factors that elicit this state, the morphological and functional alterations as well as the rationale for p53 activation are still missing. Based on literature of the past two decades, we herein summarize the evolution of the concept and provide hallmarks of nucleolar stress. Along with updated information and thorough discussion of existing confusions in the field, we pay particular attention to the current understanding of the sensing mechanisms, i.e., how stress is integrated by p53. In addition, we propose our own emphasis regarding the role of nucleolar protein NPM1 in the hallmarks of nucleolar stress and sensing mechanisms. Finally, the links of nucleolar stress to human diseases are briefly and selectively introduced.

Identification of a novel TIF-IA-NF-κB nucleolar stress response pathway.

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that artificial disruption of the PolI complex stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of NF-κB and is associated with increased nucleolar size. It is mimicked by CDK4 inhibition and is dependent upon a novel pathway involving UBF/p14ARF and S44 of the protein. We show that blocking TIF-IA degradation blocks stress effects on nucleolar size and NF-κB signalling. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, TIF-IA–NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

Nucleolar Function in Lifespan Regulation.

The nucleolus is a prominent membraneless organelle residing within the nucleus. The nucleolus has been regarded as a housekeeping structure mainly known for its role in ribosomal RNA (rRNA) production and ribosome assembly. However, accumulating evidence has revealed its functions in numerous cellular processes that control organismal physiology, thereby taking the nucleolus much beyond its conventional role in ribosome biogenesis. Perturbations in nucleolar functions have been associated with severe diseases such as cancer and progeria. Recent studies have also uncovered the role of the nucleolus in development and aging. In this review we discuss major nucleolar functions that impact organismal aging.

Dynamics and Function of Nuclear Bodies during Embryogenesis.

Nuclear bodies are RNA-rich membraneless organelles in the cell nucleus that concentrate specific sets of nuclear proteins and RNA-protein complexes. Nuclear bodies such as the nucleolus, Cajal body (CB), and the histone locus body (HLB) concentrate factors required for nuclear steps of RNA processing. Formation of these nuclear bodies occurs on genomic loci and is frequently associated with active sites of transcription. Whether nuclear body formation is dependent on a particular gene element, an active process such as transcription, or the nascent RNA present at gene loci is a topic of debate. Recently, this question has been addressed through studies in model organisms and their embryos. The switch from maternally provided RNA and protein to zygotic gene products in early embryos has been well characterized in a variety of organisms. This process, termed maternal-to-zygotic transition, provides an excellent model for studying formation of nuclear bodies before, during, and after the transcriptional activation of the zygotic genome. Here, we review findings in embryos that reveal key principles in the study of the formation and function of nucleoli, CBs, and HLBs. We propose that while particular gene elements may contribute to formation of these nuclear bodies, active transcription promotes maturation of nuclear bodies and efficient RNA processing within them.

The Multiple Functions of the Nucleolus in Plant Development, Disease and Stress Responses.

The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribose)polymerase (PARP), which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses.

Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

SUMMARYRibosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins

ABSTRACTMyosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

Prohibitin 2 localizes in nucleolus to regulate ribosomal RNA transcription and facilitate cell proliferation in RD cells

Prohibitin 2 (PHB2), as a conserved multifunctional protein, is traditionally localized in the mitochondrial inner membrane and essential for maintenance of mitochondrial function. Here, we investigated the role of PHB2 in human rhabdomyosarcoma (RMS) RD cells and found substantial localization of PHB2 in the nucleolus. We demonstrated that PHB2 knockdown inhibited RD cell proliferation through inducing cell cycle arrest and suppressing DNA synthesis. Meanwhile, down-regulation of PHB2 also induced apoptosis and promoted differentiation in fractions of RD cells. In addition, PHB2 silencing led to altered nucleolar morphology, as observed by transmission electron microscopy, and impaired nucleolar function, as evidenced by down-regulation of 45S and 18S ribosomal RNA synthesis. Consistently, upon PHB2 knockdown, occupancy of c-Myc at the ribosomal DNA (rDNA) promoter was attenuated, while more myoblast determination protein 1 (MyoD) molecules bound to the rDNA promoter. In conclusion, our findings suggest that nucleolar PHB2 is involved in maintaining nucleolar morphology and function in RD cells by regulating a variety of transcription factors, which is likely to be one of the underlying mechanisms by which PHB2 promotes tumor proliferation and represses differentiation. Our study provides new insight into the pathogenesis of RMS and novel characterizations of the highly conserved PHB2 protein.

Nucleolar stress: is there a reverse version?

The nucleolus is a dynamic structure that has roles in various physiological and pathophysiological processes. Perturbations on many aspects of the nucleolar functions are thought to cause “nucleolar stress”, which occurs in response to a variety of chemotherapeutic drugs. The main characteristic changes of nucleolar stress include: 1) reduction of the size and volume of the nucleolus; 2) inhibition of RNA Pol I-mediated rRNA synthesis; and 3) nucleoplasmic translocation of nucleolar stress-related proteins. In studying the apoptosis-inducing effect of the natural compound lovastatin (LV) on breast cancer stem cells, we unexpectedly uncovered a novel form of nucleolar stress, which we call “reverse nucleolar stress”. In our system, the canonical nucleolus stress inducer doxorubicin caused nucleoplasmic translocation of the nucleolar protein NPM and complete abolishment of Nolc1, an NPM-interacting protein and an activator of rRNA transcription. In contrast, the reverse nucleolar stress induced by LV is manifested as a more localized perinucleolar distribution of NPM and an increase in the protein level of Nolc1. Furthermore, translocation of the ribosomal protein RPL3 from the cytoplasm to the nucleolus and increased AgNOR staining were observed. These changes characterize a novel pattern of nucleolar stress doubtlessly distinguishable from the canonical one. The functional consequences of reverse nucleolar stress are not clear at present but may presumably be related to cell death or even normalization of the stressed cell. The discovery of reverse nucleolar stress opens up a new area of research in molecular and cellular biology and might have important implications in cancer therapy.