Showing papers on "Nucleolus published in 2020"

Nucleolar RNA polymerase II drives ribosome biogenesis.

Abstract: Proteins are manufactured by ribosomes—macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as ‘red laser’). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease. RNA polymerase II has an unexpected function in the nucleolus, helping to drive the expression of ribosomal RNA and to protect nucleolar structure through a mechanism involving triplex R-loop structures.

RNA-GPS Predicts SARS-CoV-2 RNA Residency to Host Mitochondria and Nucleolus.

Abstract: SARS-CoV-2 genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery Subcellular localization of its viral RNA could, thus, play important roles in viral replication and host antiviral immune response We perform computational modeling of SARS-CoV-2 viral RNA subcellular residency across eight subcellular neighborhoods We compare hundreds of SARS-CoV-2 genomes with the human transcriptome and other coronaviruses We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched toward the host mitochondrial matrix and nucleolus, and that the 5' and 3' viral untranslated regions contain the strongest, most distinct localization signals We interpret the mitochondrial residency signal as an indicator of intracellular RNA trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus A record of this paper's Transparent Peer Review process is included in the Supplemental Information

Targeting the RNA Polymerase I Transcription for Cancer Therapy Comes of Age

Abstract: Transcription of the ribosomal RNA genes (rDNA) that encode the three largest ribosomal RNAs (rRNA), is mediated by RNA Polymerase I (Pol I) and is a key regulatory step for ribosomal biogenesis. Although it has been reported over a century ago that the number and size of nucleoli, the site of ribosome biogenesis, are increased in cancer cells, the significance of this observation for cancer etiology was not understood. The realization that the increase in rRNA expression has an active role in cancer progression, not only through increased protein synthesis and thus proliferative capacity but also through control of cellular check points and chromatin structure, has opened up new therapeutic avenues for the treatment of cancer through direct targeting of Pol I transcription. In this review, we discuss the rational of targeting Pol I transcription for the treatment of cancer; review the current cancer therapeutics that target Pol I transcription and discuss the development of novel Pol I-specific inhibitors, their therapeutic potential, challenges and future prospects.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

Abstract: RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

snoRNPs: Functions in Ribosome Biogenesis.

Abstract: Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2′-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.

Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder.

Abstract: The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

Abstract: The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

Abstract: Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

Transcriptional suppression of ribosomal DNA with phase separation.

Abstract: The nucleolus is a nuclear body with multiphase liquid droplets for ribosomal RNA (rRNA) transcription. How rRNA transcription is regulated in the droplets remains unclear. Here, using single-molecule tracking of RNA polymerase I (Pol I) and chromatin-bound upstream binding factor (UBF), we reveal suppression of transcription with phase separation. For transcription, active Pol I formed small clusters/condensates that constrained rDNA chromatin in the nucleolus fibrillar center (FC). Treatment with a transcription inhibitor induced Pol I to dissociate from rDNA chromatin and to move like a liquid within the nucleolar cap that transformed from the FC. Expression of a Pol I mutant associated with a craniofacial disorder inhibited transcription by competing with wild-type Pol I clusters and transforming the FC into the nucleolar cap. The cap droplet excluded an initiation factor, ensuring robust silencing. Our findings suggest a mechanism of rRNA transcription suppression via phase separation of intranucleolar molecules governed by Pol I.

Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations.

Abstract: Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

Mapping of the nucleolar proteome reveals spatiotemporal organization related to intrinsic protein disorder

Abstract: The nucleolus is essential for ribosome biogenesis, but also involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1318 nucleolar proteins were identified; 287 localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a unique proteome composition in this phase-separated region. We identified 65 nucleolar proteins (36 unknown) to relocate to the chromosomal periphery during mitosis. Interestingly, we here observed a temporal partitioning into two phenotypes; early (prometaphase), or late (after metaphase) recruitment, suggesting phase specific functions. We further show that expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for an organelle, and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins, indicating that the perichromosomal layer is liquid-like. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

Nucleolar proteins of rat liver and Walker tumor.

Abstract: Summary Large scale preparations of highly purified nucleoli have been used for isolation of nucleolar histones, nucleolar proteins soluble in 0.14 m NaCl, and residual nucleolar proteins. Histones accounted for approximately ⅓ of liver nucleolar proteins and ⅕ of tumor nucleolar proteins. The residual nucleolar proteins comprised 60% of the tumor nucleolar proteins and 40% of liver nucleolar proteins. The starch gel electrophoretic patterns and the amino acid analyses of nucleolar and nuclear histones of both the Walker tumor and the liver were not significantly different. Although no significant differences were detected in the amino acid composition of the residual nucleolar proteins of liver and tumor nucleoli, some quantitative differences were found in the amino acid composition of the proteins soluble in 0.14 m NaCl.

Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus.

Abstract: Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.

Long Non-Coding Small Nucleolar RNA Host Genes (SNHGs) in Endocrine-Related Cancers.

Abstract: Long non-coding RNAs (lncRNAs) are emerging regulators of a diverse range of biological processes through various mechanisms. Genome-wide association studies of tumor samples have identified several lncRNAs, which act as either oncogenes or tumor suppressors in various types of cancers. Small nucleolar RNAs (snoRNAs) are predominantly found in the nucleolus and function as guide RNAs for the processing of transcription. As the host genes of snoRNAs, lncRNA small nucleolar RNA host genes (SNHGs) have been shown to be abnormally expressed in multiple cancers and can participate in cell proliferation, tumor progression, metastasis, and chemoresistance. Here, we review the biological functions and emerging mechanisms of SNHGs involved in the development and progression of endocrine-related cancers including thyroid cancer, breast cancer, pancreatic cancer, ovarian cancer and prostate cancer.

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization.

Abstract: The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

The Nucleolus and PARP1 in Cancer Biology.

Abstract: The nucleolus has been known for a long time to fulfill crucial functions in ribosome biogenesis, of which cancer cells can become addicted to in order to produce sufficient amounts of proteins for cell proliferation Recently, the nucleolus has emerged as a central regulatory hub in many other cancer-relevant processes, including stress sensing, DNA damage response, cell cycle control, and proteostasis This fostered the idea that nucleolar processes can be exploited in cancer therapy Interestingly, a significant proportion of poly(ADP-ribose) polymerase 1 (PARP1) molecules are localized in the nucleolus and PARP1 also plays crucial roles in many processes that are important in cancer biology, including genome maintenance, replication, transcription, and chromatin remodeling Furthermore, during the last years, PARP1 came into focus in oncology since it represents a promising target of pharmacological PARP inhibitors in various types of cancers Here, we provide an overview of our current understanding on the role of PARP1 in nucleolar functions and discuss potential implications in cancer biology and therapy

RNA-GPS Predicts SARS-CoV-2 RNA Localization to Host Mitochondria and Nucleolus

Abstract: The SARS-CoV-2 coronavirus is driving a global pandemic, but its biological mechanisms are less well understood. SARS-CoV-2 is an RNA virus whose multiple genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery, located across distinct cytotopic locations. Subcellular localization of its viral RNA could play important roles in viral replication and host antiviral immune response. Here we perform computational modeling of SARS-CoV-2 viral RNA localization across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes to the human transcriptome and other coronaviruses and perform systematic sub-sequence analyses to identify the responsible signals. Using state-of-the-art machine learning models, we predict that the SARS-CoV-2 RNA genome and all sgRNAs are enriched in the host mitochondrial matrix and nucleolus. The 5' and 3' viral untranslated regions possess the strongest and most distinct localization signals. We discuss the mitochondrial localization signal in relation to the formation of double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Abstract: Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH. The RRP7A a gene is involved in ribosome biogenesis. Here the authors report a homozygous missense mutation segregating with primary microcephaly, and show that this occurs via functional defects in both nucleoli and primary cilia disrupting cell proliferation and neurogenesis.

Ribosome Biogenesis Alterations in Colorectal Cancer.

Abstract: Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA

Abstract: Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.

Nucleophosmin in Its Interaction with Ligands.

Abstract: Nucleophosmin (NPM1) is a mainly nucleolar protein that shuttles between nucleoli, nucleoplasm and cytoplasm to fulfill its many functions. It is a chaperone of both nucleic acids and proteins and plays a role in cell cycle control, centrosome duplication, ribosome maturation and export, as well as the cellular response to a variety of stress stimuli. NPM1 is a hub protein in nucleoli where it contributes to nucleolar organization through heterotypic and homotypic interactions. Furthermore, several alterations, including overexpression, chromosomal translocations and mutations are present in solid and hematological cancers. Recently, novel germline mutations that cause dyskeratosis congenita have also been described. This review focuses on NPM1 interactions and inhibition. Indeed, the list of NPM1 binding partners is ever-growing and, in recent years, many studies contributed to clarifying the structural basis for NPM1 recognition of both nucleic acids and several proteins. Intriguingly, a number of natural and synthetic ligands that interfere with NPM1 interactions have also been reported. The possible role of NPM1 inhibitors in the treatment of multiple cancers and other pathologies is emerging as a new therapeutic strategy.

Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation.

Abstract: Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Interestingly, in SG assembly mutant cells (G3BP1/2ΔΔ), TDP-43 is enriched in nucleoli. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.

The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals

Abstract: The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.

Triptolide interrupts rRNA synthesis and induces the RPL23‑MDM2‑p53 pathway to repress lung cancer cells.

Abstract: Lung cancer has one of the highest mortalities of any cancer worldwide. Triptolide (TP) is a promising tumor suppressor extracted from the Chinese herb Tripterygium wilfordii. Our previous proteomics analysis revealed that TP significantly interfered with the ribosome biogenesis pathway; however, the underlying molecular mechanism remains poorly understood. The aim of the present study was to determine the molecular mechanism of TP's anticancer effect by investigating the association between ribosomal stress and p53 activation. It was found that TP induces nucleolar disintegration together with RNA polymerase I (Pol I) and upstream binding factor (UBF) translocation. TP interrupted ribosomal (r)RNA synthesis through inhibition of RNA Pol I and UBF transcriptional activation. TP treatment increased the binding of ribosomal protein L23 (RPL23) to mouse double minute 2 protein (MDM2), resulting in p53 being released from MDM2 and stabilized. Activation of p53 induced apoptosis and cell cycle arrest by enhancing the activation of p53 upregulated modulator of apoptosis, caspase 9 and caspase 3, and suppressing BCL2. In vivo experiments showed that TP significantly reduced xenograft tumor size and increased mouse body weight. Immunohistochemical assays confirmed that TP significantly increased the p53 level and induced nucleolus disintegration, during which nucleolin distribution moved from the nucleolus to the nucleoplasm, and RPL23 clustered at the edge of the cell membrane. Therefore, it was proposed that TP induces ribosomal stress, which leads to nucleolus disintegration, and inhibition of rRNA transcription and synthesis, resulting in increased binding of RPL23 with MDM2. Consequently, p53 is activated, which induces apoptosis and cell cycle arrest.

Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

Abstract: Eukaryotic chromosomes are folded into hierarchical domains, enabling the organization of the genome into functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks thought to contribute to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for genome-wide maps of nucleolar associated domains (NADs). Here we established a method based on a Dam-fused engineered nucleolar histone H2B that marks DNA contacting the nucleolus. NAD-maps of ESCs and neural progenitors revealed layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs showed higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation, chromosomes around the nucleolus acquire a more compact, rigid architecture whereas NADs specific for ESCs decrease their interaction strength within the repressive B-compartment strength, unlocking neural genes from repressive nuclear environment. The methodologies here developed will make possible to include the contribution of the nucleolus in future studies investigating the relationship between nuclear space and genome function.

Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome

Abstract: The posttranslational modification ADP-ribosylation is involved in many cellular processes, with distinct roles for poly- and mono(ADP-ribosyl)ation (PAR- and MARylation, respectively). Reversibility of intracellular MARylation was demonstrated with the discovery of MACROD1, MACROD2 and TARG1, three macrodomain-containing enzymes capable of reversing MARylation of proteins and RNA. While the three enzymes have identical activities in vitro, their roles in cells are unclear and published data are partially contradictory, possibly due to a lack of validated reagents. We developed monoclonal antibodies to study these proteins and analysed their tissue distribution and intracellular localisation. MACROD1 is most prevalent in mitochondria of skeletal muscle, MACROD2 localises to nucleo- and cytoplasm and is found so far only in neuroblastoma cells, whereas the more ubiquitously expressed TARG1 is present in nucleoplasm, nucleolus and stress granules. Loss of MACROD1 or loss of TARG1 leads to disruption of mitochondrial or nucleolar morphology, respectively, hinting at their importance for these organelles. To start elucidating the underlying mechanisms, we have mapped their interactomes using BioID. The cellular localisation of interactors supports the mitochondrial, nucleolar and stress granule localisation of MACROD1 and TARG1, respectively. Gene ontology analysis suggests an involvement of MACROD1 and TARG1 in RNA metabolism in their respective compartments. The detailed description of the hydrolases' expression, localisation and interactome presented here provides a solid basis for future work addressing their physiological function in more detail.

Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation

Abstract: Translational readthrough, ie, elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth