9-Aminoacridine Inhibits Ribosome Biogenesis by Targeting Both Transcription and Processing of Ribosomal RNA.

Aminoacridines, used for decades as antiseptic and antiparasitic agents, are prospective candidates for therapeutic repurposing and new drug development. Although the mechanisms behind their biological effects are not fully elucidated, they are most often attributed to the acridines' ability to intercalate into DNA. Here, we characterized the effects of 9-aminoacridine (9AA) on pre-rRNA metabolism in cultured mammalian cells. Our results demonstrate that 9AA inhibits both transcription of the ribosomal RNA precursors (pre-rRNA) and processing of the already synthesized pre-rRNAs, thereby rapidly abolishing ribosome biogenesis. Using a fluorescent intercalator displacement assay, we further show that 9AA can bind to RNA in vitro, which likely contributes to its ability to inhibit post-transcriptional steps in pre-rRNA maturation. These findings extend the arsenal of small-molecule compounds that can be used to block ribosome biogenesis in mammalian cells and have implications for the pharmacological development of new ribosome biogenesis inhibitors.

Cryo-EM structures of human RNA polymerase I.

RNA polymerase I (Pol I) specifically synthesizes ribosomal RNA. Pol I upregulation is linked to cancer, while mutations in the Pol I machinery lead to developmental disorders. Here we report the cryo-EM structure of elongating human Pol I at 2.7 Å resolution. In the exit tunnel, we observe a double-stranded RNA helix that may support Pol I processivity. Our structure confirms that human Pol I consists of 13 subunits with only one subunit forming the Pol I stalk. Additionally, the structure of human Pol I in complex with the initiation factor RRN3 at 3.1 Å resolution reveals stalk flipping upon RRN3 binding. We also observe an inactivated state of human Pol I bound to an open DNA scaffold at 3.3 Å resolution. Lastly, the high-resolution structure of human Pol I allows mapping of disease-related mutations that can aid understanding of disease etiology.

rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin.

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.

The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

Overexpression of ARHGAP30 suppresses growth of cervical cancer cells by downregulating ribosome biogenesis.

We aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation assays. Western-blot and q-PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP-deficient plasmid of ARHGAP30R55A . Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL.

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis.

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20-30% decreased nucleolar abundance per cell and a 30-60% downregulation of RNA Pol I transcription activity, as well as a 50-70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.

Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.

Expression of the tumor-expressed protein MageB2 enhances rRNA transcription.

An essential requirement for cells to sustain a high proliferating rate is to be paired with enhanced protein synthesis through the production of ribosomes. For this reason, part of the growth-factor signaling pathways, are devoted to activate ribosome biogenesis. Enhanced production of ribosomes is a hallmark in cancer cells, which is boosted by different mechanisms. Here we report that the nucleolar tumor-protein MageB2, whose expression is associated with cell proliferation, also participates in ribosome biogenesis. Studies carried out in both siRNA-mediated MageB2 silenced cells and CRISPR/CAS9-mediated MageB2 knockout (KO) cells showed that its expression is linked to rRNA transcription increase independently of the cell proliferation status. Mechanistically, MageB2 interacts with phospho-UBF, a protein which causes the recruitment of RNA Pol I pre-initiation complex required for rRNA transcription. In addition, cells expressing MageB2 displays enhanced phospho-UBF occupancy at the rDNA gene promoter. Proteomic studies performed in MageB2 KO cells revealed impairment in ribosomal protein (RPs) content. Functionally, enhancement in rRNA production in MageB2 expressing cells, was directly associated with an increased dynamic in protein synthesis. Altogether our results unveil a novel function for a tumor-expressed protein from the MAGE-I family. Findings reported here suggest that nucleolar MageB2 might play a role in enhancing ribosome biogenesis as part of its repertoire to support cancer cell proliferation.

Quorum sensing regulates rRNA synthesis in Saccharomyces cerevisiae.

Ribosome biogenesis requires the concerted activities of three nuclear RNA polymerases, (Pols) I, II, and III, to produce 25S, 18S and 5.8S ribosomal RNA (rRNA), messenger RNA (mRNA) encoding ribosomal proteins, and the 5S rRNA, respectively. The rRNA is processed and modified before being assembled with ribosomal proteins to produce a ribosome. Ribosome biogenesis requires extensive energetic investment by the cell, so it is critical that this process is tightly regulated in accord with cellular growth potential. Previous work revealed that rRNA synthesis in Saccharomyces cerevisiae is repressed prior to the cells shift from active growth (log phase) to limited/static growth (stationary phase). The mechanism(s) by which cells anticipate imminent stationary phase are unknown. In this study, we demonstrate that growing cells produce one or more compounds that accumulate in the growth medium, and that this compound induces repression of rRNA synthesis. When cells encounter this compound, rRNA synthesis is rapidly repressed. We further show that subunits of Pols I and II are degraded during the transition from log to stationary phase growth, but this degradation does not account for the observed repression of rRNA synthesis. Interestingly, repression of rRNA synthesis by spent media requires the nuclear exosome, implying that spent media stimulates rapid rRNA degradation. Together, these data suggest that yeast use quorum sensing to regulate rRNA synthesis in anticipation of high cell density in stationary phase.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis.

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.

Ribosome Biogenesis Alterations in Colorectal Cancer.

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.

The rRNA synthesis inhibitor CX-5461 may induce autophagy that inhibits anticancer drug-induced cell damage to leukemia cells.

Autophagy induced in cancer cells during chemotherapy is classified into two types, which differ depending on the kind of cells or anticancer drugs. The first type of autophagy contributes to the death of cells treated with drugs. In contrast, the second type plays a crucial role in preventing anticancer drug-induced cell damages; the use of an autophagy inhibitor is considered effective in improving the efficacy of chemotherapy. Thus, it is important to determine which type of autophagy is induced during chemotherapy. Here, we showed that a novel inhibitor of RNA polymerase I, suppresses growth, induces cell cycle arrest and promotes apoptosis in leukemia cell lines. The number of apoptotic cells induced by co-treatment with CX-5461 and chloroquine, an autophagy inhibitor, increased compared with CX-5461 alone. Thus, the autophagy which may be induced by CX-5461 was the second type.

NHP2 deficiency impairs rRNA biogenesis and causes pulmonary fibrosis and Høyeraal-Hreidarsson syndrome.

Telomeres are nucleoprotein structures at the end of chromosomes. The telomerase complex, constituted of the catalytic subunit TERT, the RNA matrix hTR and several cofactors, including the H/ACA box ribonucleoproteins Dyskerin, NOP10, GAR1, NAF1 and NHP2, regulates telomere length. In humans, inherited defects in telomere length maintenance are responsible for a wide spectrum of clinical premature aging manifestations including pulmonary fibrosis (PF), dyskeratosis congenita (DC), bone marrow failure and predisposition to cancer. NHP2 mutations have been so far reported only in two patients with DC. Here, we report the first case of Høyeraal-Hreidarsson syndrome, the severe form of DC, caused by biallelic missense mutations in NHP2. Additionally, we identified three unrelated patients with PF carrying NHP2 heterozygous mutations. Strikingly, one of these patients acquired a somatic mutation in the promoter of TERT that likely conferred a selective advantage in a subset of blood cells. Finally, we demonstrate that a functional deficit of human NHP2 affects ribosomal RNA biogenesis. Together, our results broaden the functional consequences and clinical spectrum of NHP2 deficiency.

Identification of distinct maturation steps involved in human 40S ribosomal subunit biosynthesis.

Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.

Non-Coding RNA-Driven Regulation of rRNA Biogenesis.

Ribosomal RNA (rRNA) biogenesis takes place in the nucleolus, the most prominent condensate of the eukaryotic nucleus. The proper assembly and integrity of the nucleolus reflects the accurate synthesis and processing of rRNAs which in turn, as major components of ribosomes, ensure the uninterrupted flow of the genetic information during translation. Therefore, the abundant production of rRNAs in a precisely functional nucleolus is of outmost importance for the cell viability and requires the concerted action of essential enzymes, associated factors and epigenetic marks. The coordination and regulation of such an elaborate process depends on not only protein factors, but also on numerous regulatory non-coding RNAs (ncRNAs). Herein, we focus on RNA-mediated mechanisms that control the synthesis, processing and modification of rRNAs in mammals. We highlight the significance of regulatory ncRNAs in rRNA biogenesis and the maintenance of the nucleolar morphology, as well as their role in human diseases and as novel druggable molecular targets.

Potential interaction of inflammatory hyperemia and hyperphosphatemia in tumorigenesis.

The present article proposes that the association of inflammation with cancer is potentially mediated by the interaction of inflammatory hyperemia and hyperphosphatemia. Hyperemia increases blood flow rate and blood volume, and hyperphosphatemia is caused by elevated serum levels of dysregulated inorganic phosphate. It is hypothesized that the interaction of inflammatory hyperemia and hyperphosphatemia circulates increased amounts of inorganic phosphate to the tumor microenvironment, where increased uptake of inorganic phosphate through sodium-phosphate cotransporters is sequestered in cells. Elevated levels of intracellular phosphorus increase biosynthesis of ribosomal RNA, leading to increased protein synthesis that supports tumor growth. The present article also proposes that the interaction of inflammatory hyperemia and hyperphosphatemia may help explain a chemopreventive mechanism associated with NSAIDs.

Replicative senescence is distinguishable from DNA damage-induced senescence by increased methylation of promoter of rDNA and reduced expression of rRNA.

Human fibroblasts become senescent after a limited number of replications or by diverse stresses, such as DNA damage. However, replicative and damage induced senescence are indistinguishable in respect to proliferation cessation and expression of senescence markers, senescence-associated ß-galactosidase, p16 and p21. Here, we show that senescence types can be distinguished by reduced levels of 18S, 5.8S and 28S rRNA, in replicative but not induced senescence. We also demonstrate that promoter region of rRNA is hypermethylated in replicative senescence. The findings show that expression level of rRNA or methylation of its promoter can be used to distinguish between senescence types.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.