Free ribosomal proteins as culprits for nucleolar stress.

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.

SnapShot: Eukaryotic ribosome biogenesis II.

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.

Subverting the Canon: Novel Cancer-Promoting Functions and Mechanisms for snoRNAs.

Small nucleolar RNAs (snoRNAs) constitute a class of intron-derived non-coding RNAs ranging from 60 to 300 nucleotides. Canonically localized in the nucleolus, snoRNAs play a pivotal role in RNA modifications and pre-ribosomal RNA processing. Based on the types of modifications they involve, such as methylation and pseudouridylation, they are classified into two main families-box C/D and H/ACA snoRNAs. Recent investigations have revealed the unconventional synthesis and biogenesis strategies of snoRNAs, indicating their more profound roles in pathogenesis than previously envisioned. This review consolidates recent discoveries surrounding snoRNAs and provides insights into their mechanistic roles in cancer. It explores the intricate interactions of snoRNAs within signaling pathways and speculates on potential therapeutic solutions emerging from snoRNA research. In addition, it presents recent findings on the long non-coding small nucleolar RNA host gene (lncSNHG), a subset of long non-coding RNAs (lncRNAs), which are the transcripts of parental SNHGs that generate snoRNA. The nucleolus, the functional epicenter of snoRNAs, is also discussed. Through a deconstruction of the pathways driving snoRNA-induced oncogenesis, this review aims to serve as a roadmap to guide future research in the nuanced field of snoRNA-cancer interactions and inspire potential snoRNA-related cancer therapies.

Polyamine Dysregulation and Nucleolar Disruption in Alzheimer's Disease.

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

ZNF692 regulates nucleolar morphology by interacting with NPM1 and modifying its self-assembly properties.

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.

Rna Buffering Fluorogenic Probe for Nucleolar Morphology Stable Imaging And Nucleolar Stress-Generating Agents Screening.

In the realm of cell research, membraneless organelles have become a subject of increasing interest. However, their ever-changing and amorphous morphological characteristics have long presented a formidable challenge when it comes to studying their structure and function. In this paper, a fluorescent probe Nu-AN is reported, which exhibits the remarkable capability to selectively bind to and visualize the nucleolus morphology, the largest membraneless organelle within the nucleus. Nu-AN demonstrates a significant enhancement in fluorescence upon its selective binding to nucleolar RNA, due to the inhibited twisted intramolecular charge-transfer (TICT) and reduced hydrogen bonding with water. What sets Nu-AN apart is its neutral charge and weak interaction with nucleolus RNA, enabling it to label the nucleolus selectively and reversibly. This not only reduces interference but also permits the replacement of photobleached probes with fresh ones outside the nucleolus, thereby preserving imaging photostability. By closely monitoring morphology-specific changes in the nucleolus with this buffering fluorogenic probe, screenings for agents are conducted that induce nucleolar stress within living cells.

Dynamic nucleolar phase separation influenced by non-canonical function of LIN28A instructs pluripotent stem cell fate decisions.

LIN28A is important in somatic reprogramming and pluripotency regulation. Although previous studies addressed that LIN28A can repress let-7 microRNA maturation in the cytoplasm, few focused on its role within the nucleus. Here, we show that the nucleolus-localized LIN28A protein undergoes liquid-liquid phase separation (LLPS) in mouse embryonic stem cells (mESCs) and in vitro. The RNA binding domains (RBD) and intrinsically disordered regions (IDR) of LIN28A contribute to LIN28A and the other nucleolar proteins' phase-separated condensate establishment. S120A, S200A and R192G mutations in the IDR result in subcellular mislocalization of LIN28A and abnormal nucleolar phase separation. Moreover, we find that the naive-to-primed pluripotency state conversion and the reprogramming are associated with dynamic nucleolar remodeling, which depends on LIN28A's phase separation capacity, because the LIN28A IDR point mutations abolish its role in regulating nucleolus and in these cell fate decision processes, and an exogenous IDR rescues it. These findings shed light on the nucleolar function in pluripotent stem cell states and on a non-canonical RNA-independent role of LIN28A in phase separation and cell fate decisions.

The perinucleolar compartment: structure, function, and utility in anti-cancer drug development.

The perinucleolar compartment (PNC) was initially identified as a nuclear structure enriched for the polypyrimidine tract-binding protein. Since then, the PNC has been implicated in carcinogenesis. The prevalence of this compartment is positively correlated with disease progression in various types of cancer, and its expression in primary tumors is linked to worse patient outcomes. Using the PNC as a surrogate marker for anti-cancer drug efficacy has led to the development of a clinical candidate for anti-metastasis therapies. The PNC is a multicomponent nuclear body situated at the periphery of the nucleolus. Thus far, several non-coding RNAs and RNA-binding proteins have been identified as the PNC components. Here, we summarize the current understanding of the structure and function of the PNC, as well as its recurrent links to cancer progression and metastasis.

Multiscale biophysical analysis of nucleolus disassembly during mitosis.

During cell division, precise and regulated distribution of cellular material between daughter cells is a critical step and is governed by complex biochemical and biophysical mechanisms. To achieve this, membraneless organelles and condensates often require complete disassembly during mitosis. The biophysical principles governing the disassembly of condensates remain poorly understood. Here, we used a physical biology approach to study how physical and material properties of the nucleolus, a prominent nuclear membraneless organelle in eukaryotic cells, change during mitosis and across different scales. We found that nucleolus disassembly proceeds continuously through two distinct phases with a slow and reversible preparatory phase followed by a rapid irreversible phase that was concurrent with the nuclear envelope breakdown. We measured microscopic properties of nucleolar material including effective diffusion rates and binding affinities as well as key macroscopic properties of surface tension and bending rigidity. By incorporating these measurements into the framework of critical phenomena, we found evidence that near mitosis surface tension displays a power-law behavior as a function of biochemically modulated interaction strength. This two-step disassembly mechanism maintains structural and functional stability of nucleolus while enabling its rapid and efficient disassembly in response to cell cycle cues.

New Functional Motifs for the Targeted Localization of Proteins to the Nucleolus in Drosophila and Human Cells.

The nucleolus is a significant nuclear organelle that is primarily known for its role in ribosome biogenesis. However, emerging evidence suggests that the nucleolus may have additional functions. Particularly, it is involved in the organization of the three-dimensional structure of the genome. The nucleolus acts as a platform for the clustering of repressed chromatin, although this process is not yet fully understood, especially in the context of Drosophila. One way to study the regions of the genome that cluster near the nucleolus in Drosophila demands the identification of a reliable nucleolus-localizing signal (NoLS) motif(s) that can highly specifically recruit the protein of interest to the nucleolus. Here, we tested a series of various NoLS motifs from proteins of different species, as well as some of their combinations, for the ability to drive the nucleolar localization of the chimeric H2B-GFP protein. Several short motifs were found to effectively localize the H2B-GFP protein to the nucleolus in over 40% of transfected Drosophila S2 cells. Furthermore, it was demonstrated that NoLS motifs derived from Drosophila proteins exhibited greater efficiency compared to that of those from other species.

Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression.

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.

Intrinsic disorder of a nucleoplasmin-like histone chaperone specifies its discrete nuclear and nucleolar functions.

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.

Reprogramming nucleolar size by genetic perturbation of the extranuclear Rab GTPases Ypt6 and Ypt32.

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.

Nucleolus imaging based on naphthalimide derivatives.

Nucleolus was an important cellular organelle. The abnormal morphology and number of the nucleolus have been considered as diagnostic biomarkers for some human diseases. However, the imaging agent based on nucleolus was limited. In this manuscript, a series of nucleolar fluorescent probes based on naphthalimide derivatives (NI-1 âˆ¼ NI-5) had been designed and synthesized. NI-1 âˆ¼ NI-5 could penetrate cell membranes and nuclear membranes, achieve clear nucleolar staining in living cells. These results suggested that the presence of amino groups on the side chains of naphthalimide backbone could enhance the targeting to the cell nucleolus. In addition, the molecular docking results showed that NI-1 âˆ¼ NI-5 formed hydrogen bonds and hydrophobic interactions with RNA, and exhibited enhanced fluorescence upon binding with RNA. These results will provide favorable support for the diagnosis and treatment of nucleolus-related diseases in the future.

GPATCH4 contributes to nucleolus morphology and its dysfunction impairs cell viability.

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated ß-galactosidase (SA-ß-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability.

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

Distinct states of nucleolar stress induced by anticancer drugs.

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed 'nucleolar normality score' was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I- recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Ribosomes are cell structures within a compartment called the nucleolus that are required to make proteins, which are essential for cell function. Due to their uncontrolled growth and division, cancer cells require many proteins and therefore have a particularly high demand for ribosomes. Due to this, some anti-cancer drugs deliberately target the activities of the nucleolus. However, it was not clear if anti-cancer drugs with other targets also disrupt the nucleolus, which may result in side effects. Previously, it had been difficult to study how nucleoli work, partly because in human cells they vary naturally in shape, size, and number. Potapova et al. used fluorescent microscopy to develop a new way of assessing nucleoli based on the location and ratio of certain proteins. These measurements were used to calculate a "nucleolar normality score". Potapova et al. then tested over a thousand anti-cancer drugs in healthy and cancerous human cells. Around 10% of the tested drugs changed the nucleolar normality score when compared to placebo treatment, indicating that they caused nucleolar stress. For most of these drugs, the nucleolus was not the intended target, suggesting that disrupting it was an unintended side effect. Drugs inhibiting proteins called cyclin-dependent kinases caused the most drastic changes in the size and shape of nucleoli, disrupting them completely. These kinases are known to be involved in activating enzymes required for general transcription. Potapova et al. showed that they also are involved in production of ribosomal RNA, revealing an additional role in coordinating ribosome assembly. Taken together, the findings suggest that evaluating the effect of new anti-cancer drugs on the nucleolus could help to develop future treatments with less toxic side effects. The experiments also reveal new avenues for researching how cyclin-dependent kinases control the production of RNA more generally.

Nucleolar reorganization after cellular stress is orchestrated by SMN shuttling between nuclear compartments.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1.

The Effects of Deregulated Ribosomal Biogenesis in Cancer.

Ribosomes are macromolecular ribonucleoprotein complexes assembled from RNA and proteins. Functional ribosomes arise from the nucleolus, require ribosomal RNA processing and the coordinated assembly of ribosomal proteins (RPs), and are frequently hyperactivated to support the requirement for protein synthesis during the self-biosynthetic and metabolic activities of cancer cells. Studies have provided relevant information on targeted anticancer molecules involved in ribosome biogenesis (RiBi), as increased RiBi is characteristic of many types of cancer. The association between unlimited cell proliferation and alterations in specific steps of RiBi has been highlighted as a possible critical driver of tumorigenesis and metastasis. Thus, alterations in numerous regulators and actors involved in RiBi, particularly in cancer, significantly affect the rate and quality of protein synthesis and, ultimately, the transcriptome to generate the associated proteome. Alterations in RiBi in cancer cells activate nucleolar stress response-related pathways that play important roles in cancer-targeted interventions and immunotherapies. In this review, we focus on the association between alterations in RiBi and cancer. Emphasis is placed on RiBi deregulation and its secondary consequences, including changes in protein synthesis, loss of RPs, adaptive transcription and translation, nucleolar stress regulation, metabolic changes, and the impaired ribosome biogenesis checkpoint.