ABSTRACT
In eukaryotes, the nucleolus is the critical non-membranous organelle within nuclei that is responsible for ribosomal DNA (rDNA) transcription and ribosome biogenesis. The transcription of rDNA, a rate-limiting step for ribosome biogenesis, is tightly regulated to meet the demand for global protein synthesis in response to cell physiology, especially in neurons, which undergo rapid changes in morphology and protein composition during development and synaptic plasticity. However, it is unknown how the pre-initiation complex for rDNA transcription is efficiently assembled within the nucleolus in neurons. Here, we report that the nucleolar protein, coronin 2B, regulates rDNA transcription and maintains nucleolar function through direct interaction with upstream binding factor (UBF), an activator of RNA polymerase I transcriptional machinery. We show that coronin 2B knockdown impairs the formation of the transcription initiation complex, inhibits rDNA transcription, destroys nucleolar integrity, and ultimately induces nucleolar stress. In turn, coronin 2B-mediated nucleolar stress leads to p53 stabilization and activation, eventually resulting in neuronal apoptosis. Thus, we identified that coronin 2B coordinates with UBF to regulate rDNA transcription and maintain proper nucleolar function in neurons.
Subject(s)
Apoptosis , Cell Nucleolus , Neurons , Pol1 Transcription Initiation Complex Proteins , Apoptosis/genetics , Cell Nucleolus/metabolism , Neurons/metabolism , Animals , Pol1 Transcription Initiation Complex Proteins/metabolism , Pol1 Transcription Initiation Complex Proteins/genetics , Humans , DNA, Ribosomal/metabolism , DNA, Ribosomal/genetics , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Mice , Stress, PhysiologicalABSTRACT
Despite tremendous advances in the diagnosis and treatment of NSCLC, the morbidity and mortality of NSCLC still rank high worldwide. Epithelial-mesenchymal transition (EMT) is vital to the invasion, metastasis, and recurrence of NSCLC. Unfortunately, the mechanism behind NSCLC cancer cell EMT remains elusive. Therefore, determining the potential key molecules that induce EMT is important. TATA-binding protein-associated factor-1 (TAF1) is an important component of the preinitiation complex (PIC) that is dysregulated in carcinogenesis. However, the role of TAF1 in NSCLC development is unknown. Therefore, we studied the role of TAF1 in the pathogenesis of NSCLC. First, the expression of TAF1 was determined in human NSCLC tissues and cell lines. TAF1-overexpressing and TAF1 knockdown cell lines were established to evaluate the effect of TAF1 on NSCLC cell proliferation, invasion and migration by colony formation and Transwell assays. The target genes of TAF1 were identified by PCR array and verified by luciferase reporter assay. Our data demonstrated that TAF1 is upregulated in NSCLC. Higher TAF1 expression predicted poor outcomes in NSCLC patients. Mechanistically, TAF1 transcriptionally activated TGFß1, thus promoting NSCLC cell EMT and the development of NSCLC. Targeting TAF1/TGFß1 signalling may be potentially helpful as a therapeutic for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Pol1 Transcription Initiation Complex Proteins , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , Neoplasm Invasiveness/genetics , Pol1 Transcription Initiation Complex Proteins/geneticsABSTRACT
Oncogenic alterations underlying B-cell acute lymphoblastic leukemia (B-ALL) in adults remain incompletely elucidated. To uncover novel oncogenic drivers, we performed RNA sequencing and whole-genome analyses in a large cohort of unresolved B-ALL. We identified a novel subtype characterized by a distinct gene expression signature and the unique association of 2 genomic microdeletions. The 17q21.31 microdeletion resulted in a UBTF::ATXN7L3 fusion transcript encoding a chimeric protein. The 13q12.2 deletion resulted in monoallelic ectopic expression of the homeobox transcription factor CDX2, located 138 kb in cis from the deletion. Using 4C-sequencing and CRISPR interference experiments, we elucidated the mechanism of CDX2 cis-deregulation, involving PAN3 enhancer hijacking. CDX2/UBTF ALL (n = 26) harbored a distinct pattern of additional alterations including 1q gain and CXCR4 activating mutations. Within adult patients with Ph- B-ALL enrolled in GRAALL trials, patients with CDX2/UBTF ALL (n = 17/723, 2.4%) were young (median age, 31 years) and dramatically enriched in females (male/female ratio, 0.2, P = .002). They commonly presented with a pro-B phenotype ALL and moderate blast cell infiltration. They had poor response to treatment including a higher risk of failure to first induction course (19% vs 3%, P = .017) and higher post-induction minimal residual disease (MRD) levels (MRD ≥ 10-4, 93% vs 46%, P < .001). This early resistance to treatment translated into a significantly higher cumulative incidence of relapse (75.0% vs 32.4%, P = .004) in univariate and multivariate analyses. In conclusion, we discovered a novel B-ALL entity defined by the unique combination of CDX2 cis-deregulation and UBTF::ATXN7L3 fusion, representing a high-risk disease in young adults.
Subject(s)
CDX2 Transcription Factor , Pol1 Transcription Initiation Complex Proteins , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Transcription Factors , Adult , CDX2 Transcription Factor/genetics , Female , Genes, Homeobox , Humans , Male , Neoplasm, Residual/genetics , Oncogene Proteins, Fusion , Pol1 Transcription Initiation Complex Proteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Transcription Factors/geneticsABSTRACT
Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.
Subject(s)
Pol1 Transcription Initiation Complex Proteins , RNA Polymerase I , Animals , Child , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Humans , Mice , Nucleosomes , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase I/genetics , RNA, Ribosomal/genetics , Transcription, GeneticABSTRACT
Senescence of smooth muscle cells (SMCs) has a crucial role in the pathogenesis of abdominal aortic aneurysm (AAA), a disease of vascular degeneration. Perturbation of cellular ribosomal DNA (rDNA) transcription triggers nucleolar stress response. Previously we demonstrated that induction of nucleolar stress in SMCs elicited cell cycle arrest via the ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR)-p53 axis. However, the specific roles of nucleolar stress in vascular degeneration remain unexplored. In the present study, we demonstrated for the first time that in both human and animal AAA tissues, there were non-coordinated changes in the expression of RNA polymerase I machinery components, including a downregulation of transcription initiation factor-IA (TIF-IA). Genetic deletion of TIF-IA in SMCs in mice (smTIF-IA-/-) caused spontaneous aneurysm-like lesions in the aorta. In vitro, induction of nucleolar stress triggered a non-canonical DNA damage response, leading to p53 phosphorylation and a senescence-like phenotype in SMCs. In human AAA tissues, there was increased nucleolar stress in medial cells, accompanied by localized DNA damage response within the nucleolar compartment. Our data suggest that perturbed rDNA transcription and induction of nucleolar stress contribute to the pathogenesis of AAA. Moreover, smTIF-IA-/- mice may be a novel animal model for studying spontaneous AAA-like vascular degenerations.
Subject(s)
Cell Nucleolus/pathology , Cell Proliferation , Cellular Senescence , DNA Damage , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Animals , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/metabolism , Aortic Aneurysm, Abdominal/pathology , Cell Cycle Checkpoints , Cell Line , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Disease Models, Animal , Humans , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Phenotype , Phosphorylation , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , Signal Transduction , Transcription, Genetic , Tumor Suppressor Protein p53/metabolismABSTRACT
Regulation of ribosomal transcription is under tight control from environmental stimuli, and this control involves changes in the chromatin structure. The underlying mechanism of how chromatin changes in response to nutrient and energy supply in the cell is still unclear. The chromatin-remodeling complex B-WICH is involved in activating the ribosomal transcription, and we show here that knock down of the B-WICH component WSTF results in cells that do not respond to glucose. The promoter is less accessible, and RNA pol I and its transcription factors SL1/TIF-1B and RRN3/TIF-1A, as well as the proto-oncogene c-MYC and the activating deacetylase SIRT7 do not bind upon glucose stimulation. In contrast, the repressive chromatin state that forms after glucose deprivation is reversible, and RNA pol I factors are recruited. WSTF knock down results in an accumulation of the ATPase CHD4, a component of the NuRD chromatin remodeling complex, which is responsible for establishing a repressive poised state at the promoter. The TTF-1, which binds and affect the binding of the chromatin complexes, is important to control the association of activating chromatin component UBF. We suggest that B-WICH is required to allow for a shift to an active chromatin state upon environmental stimulation, by counteracting the repressive state induced by the NuRD complex.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Glucose/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Ribosomes/genetics , Transcription, Genetic/genetics , Adenosine Triphosphatases/genetics , Cell Line , Cell Line, Tumor , DNA-Binding Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Nuclear Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/genetics , RNA Polymerase I/genetics , Sirtuins/genetics , Transcription Factors/geneticsABSTRACT
PURPOSE: DNA mismatch repair (MMR) deficiency is a hallmark of Lynch syndrome, the most common inherited cancer syndrome. MMR-deficient cancer cells accumulate numerous insertion/deletion mutations at microsatellites. Mutations of coding microsatellites (cMS) lead to the generation of immunogenic frameshift peptide (FSP) neoantigens. As the evolution of MMR-deficient cancers is triggered by mutations inactivating defined cMS-containing tumor suppressor genes, distinct FSP neoantigens are shared by most MMR-deficient cancers. To evaluate safety and immunogenicity of an FSP-based vaccine, we performed a clinical phase I/IIa trial (Micoryx). PATIENTS AND METHODS: The trial comprised three cycles of four subcutaneous vaccinations (FSP neoantigens derived from mutant AIM2, HT001, TAF1B genes) mixed with Montanide ISA-51 VG over 6 months. Inclusion criteria were history of MMR-deficient colorectal cancer (UICC stage III or IV) and completion of chemotherapy. Phase I evaluated safety and toxicity as primary endpoint (six patients), phase IIa addressed cellular and humoral immune responses (16 patients). RESULTS: Vaccine-induced humoral and cellular immune responses were observed in all patients vaccinated per protocol. Three patients developed grade 2 local injection site reactions. No vaccination-induced severe adverse events occurred. One heavily pretreated patient with bulky metastases showed stable disease and stable CEA levels over 7 months. CONCLUSIONS: FSP neoantigen vaccination is systemically well tolerated and consistently induces humoral and cellular immune responses, thus representing a promising novel approach for treatment and even prevention of MMR-deficient cancer.
Subject(s)
Antigens, Neoplasm/administration & dosage , Cancer Vaccines/administration & dosage , Colorectal Neoplasms, Hereditary Nonpolyposis/therapy , Colorectal Neoplasms/prevention & control , DNA Mismatch Repair , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/adverse effects , Antigens, Neoplasm/genetics , Cancer Vaccines/adverse effects , Cancer Vaccines/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms, Hereditary Nonpolyposis/complications , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/immunology , DNA-Binding Proteins/genetics , Female , Frameshift Mutation , Humans , Injections, Subcutaneous , Male , Microsatellite Instability , Middle Aged , Pol1 Transcription Initiation Complex Proteins/genetics , Proteins/genetics , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/adverse effects , Vaccines, Subunit/geneticsABSTRACT
OBJECTIVE: Previous studies have demonstrated that TNFAIP8 is a cancer-promoting gene. However, the role of TNFAIP8 in esophageal cancer (ECa) has not been reported. The aim of this investigation was to investigate the expression of TNFAIP8 in ECa, and to further explore whether it could regulate cisplatin resistance to this cancer via modulating TAF-Iα expression and promote malignant progression of ECa. PATIENTS AND METHODS: Quantitative Real Time-PCR (qRT-PCR) was performed to examine the expression of TNFAIP8 in 47 tumor tissue specimens and adjacent ones of ECa patients, and the interplay between TNFAIP8 expression and prognosis of patients with ECa was then analyzed. Further, qRT-PCR was applied to verify TNFAIP8 level in ECa cell lines. In addition, the TNFAIP8 knockdown model was constructed in ECa cisplatin-resistant cell lines including EC-109/DDP and OE19/DDP, and then, CCK8 and transwell assays were performed to analyze the impact of TNFAIP8 on the biological function of ECa cells; meanwhile, the Luciferase reporter gene assay and cell reverse experiments were finally conducted to explore its underlying mechanisms. RESULTS: The qRT-PCR results revealed that the TNFAIP8 level in tumor tissue samples of ECa patients was remarkably higher than that in adjacent ones, and the difference was statistically significant. Similarly, the overall survival rate of patients with high expression of TNFAIP8 was lower when compared with patients with low expression of TNFAIP8. EC-109/DDP and OE19/DDP, the ECa cisplatin-resistant cell lines, were successfully constructed; subsequently, it was found that the proliferation, invasiveness, and metastasis ability of ECa cells in TNFAIP8 knockdown group was remarkably decreased compared with those in the sh-NC group. At the same time, the Western blot results illustrated that the expression of TAF-Iαwas remarkably elevated in the TNFAIP8 knockdown group. In addition, the Luciferase reporting assay and cell reverse experiments also demonstrated that there existed a mutual regulation effect between TNFAIP8 and TAF-Iα, which might together affect the malignant progression of ECa. CONCLUSIONS: The expression of TNFAIP8 was found remarkably enhanced in ECa tissues and cell lines, which might be closely relevant to the poor prognosis of patients with ECa. Additionally, it was found that TNFAIP8 may regulate cisplatin resistance and promote malignant progression of ECa by modulating TAF-Iα expression.
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis Regulatory Proteins/biosynthesis , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Esophageal Neoplasms/metabolism , Pol1 Transcription Initiation Complex Proteins/biosynthesis , Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Cisplatin/therapeutic use , Disease Progression , Drug Resistance, Neoplasm/physiology , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/genetics , Humans , Pol1 Transcription Initiation Complex Proteins/geneticsABSTRACT
Ribosomal DNA (rDNA) transcription is a limiting step in ribosome biogenesis, crucial for protein synthesis and cell growth-especially at the early stages of embryonic development-and is regulated in a mammalian target of rapamycin (mTOR)-dependent manner. Our previous report demonstrated that treatment with mTOR inhibitors during artificial embryonic activation improved the development of embryos derived from somatic cell nuclear transfer (SCNT). We hypothesize that inhibition of ribosome biogenesis in somatic cells facilitates reactivation of embryonic nucleolar establishment and ribosome biogenesis in SCNT embryos. Herein, we show that mTOR inhibitors suppressed ribosome biogenesis in somatic cells, and more importantly, improved development potential of SCNT embryos (blastocyst rate, 34% vs 24%). SCNT embryos derived from drug-treated somatic cells exhibited higher levels of 47S, 18S, and 5S rRNAs, upstream binding factor (UBF) mRNA, ribosomal protein S6; they also improved the rebuilding of the nucleolar ultrastructure. In addition, treatment of donor cells with the RNA polymerase I (Pol I) inhibitor cx5461 caused similar effects on SCNT embryos. These results indicated that transient inhibition of rDNA transcription in donor cells facilitated the establishment of functional nucleoli and improved preimplantation development of SCNT embryos.
Subject(s)
Cell Nucleolus/genetics , DNA, Ribosomal/genetics , Embryonic Development/genetics , Ribosomes/genetics , Transcription, Genetic/genetics , Animals , Blastocyst/physiology , Cloning, Organism/methods , Embryo, Mammalian/physiology , Female , Mice , Nuclear Transfer Techniques , Organelle Biogenesis , Pol1 Transcription Initiation Complex Proteins/genetics , TOR Serine-Threonine Kinases/geneticsABSTRACT
BACKGROUND: A new monogenic neurodegenerative disease affecting ribosomal metabolism has recently been identified in association with a monoallelic UBTF putative gain of function variant (NM_001076683.1:c.628G>A, hg19). Phenotype is consistent among these probands with progressive motor, cognitive, and behavioural regression in early to middle childhood. CASE PRESENTATION: We report on a child with this monoallelic UBTF variant who presented with progressive disease including regression, episodes of subacute deterioration during febrile illnesses and a remarkable EEG pattern with a transient pattern of semi-periodic slow waves. CONCLUSIONS: This case further supports the phenotype-genotype correlation of neurodegeneration associated with UBTF c.628G>A. Moreover, it brings new insights into the clinical features and EEG that could possibly serve as diagnostic markers of this otherwise nonspecific phenotype.
Subject(s)
Neurodegenerative Diseases/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Child , Genetic Variation , Genotype , Humans , Male , PhenotypeABSTRACT
Ribosome biogenesis is critical for proliferating cells and requires the coordinated activities of three eukaryotic RNA polymerases. We recently showed that the small ubiquitin-like modifier (SUMO) system controls the global level of RNA polymerase II (Pol II)-controlled transcription in mammalian cells by regulating cyclin-dependent kinase 9 activity. Here, we present evidence that the SUMO system also plays a critical role in the control of Pol I transcription. Using an siRNA-based knockdown approach, we found that multiple SUMO E3 ligases of the PIAS (protein inhibitor of activated STAT) family are involved in SUMO-mediated repression of ribosomal DNA (rDNA) gene transcription. We demonstrate that endogenous SUMO represses rDNA transcription primarily by repressing upstream-binding factor and proto-oncogene c-Myc expression and that ectopic overexpression of SUMO-associated enzymes additionally represses rDNA transcription via c-Myc SUMOylation and its subsequent degradation. The results of our study reveal a critical role of SUMOylation in the control of rDNA transcription, uncover the underlying mechanisms involved, and indicate that the SUMO system coordinates Pol I- and Pol II-mediated transcription in mammalian cells.
Subject(s)
DNA, Ribosomal/genetics , Down-Regulation/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Proto-Oncogene Proteins c-myc/genetics , DNA, Ribosomal/metabolism , Gene Expression Profiling , HEK293 Cells , HeLa Cells , Humans , Pol1 Transcription Initiation Complex Proteins/metabolism , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/metabolism , Sumoylation , Transcriptional Activation/geneticsABSTRACT
Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30â Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.
Subject(s)
Cell Nucleolus/metabolism , Pol1 Transcription Initiation Complex Proteins/genetics , RNA, Ribosomal/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA, Ribosomal/genetics , Humans , Nucleolus Organizer Region/genetics , Nucleolus Organizer Region/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Transcription, Genetic/geneticsABSTRACT
Upstream binding transcription factor (UBTF) is a co-regulator of RNA polymerase I by constituting an initiation complex on rRNA genes. UBTF plays a role in rDNA bending and its maintenance in "open" state. It exists as two splicing variants, UBTF1 and UBTF2, which cannot be discerned with antibodies raised against UBTF. We investigated the ultrastructural localization of each variant in cells synthesizing GFP-tagged UBTF1 or UBTF2 by using anti-GFP antibodies and pre-embedding nanogold strategy. Detailed 3D distribution of UBTF1 and 2 was also studied by electron tomography. In control cells, the two isoforms are very abundant within fibrillar centers, but their repartition strongly differs. Electron tomography shows that UBTF1 is disposed as fibrils that are folded in coils whereas UBTF2 is localized homogenously, preferentially at their cortical area. As UBTF is a useful marker to trace rDNA genes, we used these data to improve our previous model of 3D organization of active transcribing rDNA gene within fibrillar centers. Finally, when rRNA synthesis is inhibited during actinomycin D treatment or entry in mitosis, UBTF1 and UBTF2 show a similar distribution along extended 3D loop-like structures. Altogether these data suggest new roles for UBTF1 and UBTF2 isoforms in the organization of active and inactive rDNA genes.
Subject(s)
Electron Microscope Tomography/methods , Pol1 Transcription Initiation Complex Proteins/metabolism , Protein Isoforms/metabolism , Protein Isoforms/ultrastructure , Cell Line , Humans , Microscopy, Fluorescence , Pol1 Transcription Initiation Complex Proteins/geneticsABSTRACT
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Subject(s)
Pol1 Transcription Initiation Complex Proteins/genetics , RNA Polymerase I/genetics , DNA, Ribosomal/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA Precursors/genetics , RNA, Ribosomal , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription, GeneticABSTRACT
The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is required for MLL fusion-driven acute myeloid leukemia (AML) through direct regulation of pro-leukemic target genes such as Hoxa9 and Meis1. However, the role of the PAF1c in normal hematopoiesis is unknown. Here, we discovered that the PAF1c subunit, CDC73, is required for both fetal and adult hematopoiesis. Loss of Cdc73 in hematopoietic cells is lethal because of extensive bone marrow failure. Cdc73 has an essential cell-autonomous role for adult hematopoietic stem cell function in vivo, and deletion of Cdc73 results in cell-cycle defects in hematopoietic progenitors. Gene expression profiling indicated a differential regulation of Hoxa9/Meis1 gene programs by CDC73 in progenitors compared with AML cells, suggesting disease-specific functions. Thus, the PAF1c subunit, CDC73 is essential for hematopoietic stem cell function but exhibits leukemia-specific regulation of self-renewal gene programs in AML cells.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Tumor Suppressor Proteins/genetics , Acute Disease , Animals , Cell Line, Tumor , Fetus/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism , Pol1 Transcription Initiation Complex Proteins/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.
Subject(s)
Cell Nucleolus/metabolism , Dystroglycans/metabolism , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA, Ribosomal/biosynthesis , Animals , Cell Proliferation/genetics , Cytoplasm/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Dystroglycans/antagonists & inhibitors , Dystroglycans/genetics , Mice , Myoblasts , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Oxidative Stress , Pol1 Transcription Initiation Complex Proteins/genetics , Protein Domains/genetics , RNA, Ribosomal/genetics , Ribosomes/metabolism , Transcription, Genetic , Up-Regulation/geneticsABSTRACT
RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , RNA Polymerase I/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Amino Acid Sequence , Cell Cycle Proteins/genetics , Conserved Sequence , Humans , Nuclear Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/chemistry , Pol1 Transcription Initiation Complex Proteins/genetics , Pol1 Transcription Initiation Complex Proteins/metabolism , RNA, Ribosomal/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion , Transcription, GeneticABSTRACT
INTRODUCTION: Neurodegenerative diseases of childhood present with progressive decline in cognitive, social, and motor function and are frequently associated with seizures in different stages of the disease. Here we report a patient with severe progressive neurodegeneration with drug-resistant epilepsy of unknown etiology from the age of 2 years. METHODS AND RESULTS: Using whole exome sequencing, we found heterozygous missense de novo variant c.628G > A (p.Glu210Lys) in the UBTF gene. This variant was recently described as de novo in 11 patients with similar neurodegeneration characterized by developmental decline initially confined to motor development followed by language regression, appearance of an extrapyramidal movement disorder, and leading to severe intellectual disability. In 3 of the 11 patients described so far, seizures were also present. CONCLUSIONS: Our report expands the complex phenotype of neurodegeneration associated with the c.628G > A variant in the UBTF gene and helps to clarify the relation between this one single recurrent pathogenic variant described in this gene to date and its phenotype. The UBTF gene should be considered a novel candidate gene in neurodegeneration with or without epilepsy.
Subject(s)
Drug Resistant Epilepsy/genetics , Mutation/genetics , Neurodegenerative Diseases/genetics , Phenotype , Pol1 Transcription Initiation Complex Proteins/genetics , Adolescent , Drug Resistant Epilepsy/complications , Drug Resistant Epilepsy/diagnostic imaging , Humans , Male , Neurodegenerative Diseases/complications , Neurodegenerative Diseases/diagnostic imagingABSTRACT
A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of [Formula: see text]. Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and Bud13 as a factor controlling the levels of ac4C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) [Formula: see text]. Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered [Formula: see text]. These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence [Formula: see text] biogenesis.
Subject(s)
Adenosine Triphosphatases/genetics , Pol1 Transcription Initiation Complex Proteins/genetics , Protein Biosynthesis , RNA, Transfer/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , TATA-Binding Protein Associated Factors/genetics , Alkyl and Aryl Transferases/genetics , Carrier Proteins/genetics , Mutation , Nuclear Pore Complex Proteins/genetics , Oxidoreductases/genetics , RNA Precursors/biosynthesis , RNA Precursors/genetics , RNA, Transfer/genetics , Saccharomyces cerevisiae/genetics , tRNA Methyltransferases/geneticsABSTRACT
Spindlin1 (SPIN1), a histone modification reader protein, was enriched in the cell nucleolus and facilitated rRNA expression. However, how SPIN1 localizes to the nucleolus and its functional role in rRNA gene expression remain unresolved. Here, we identified a nucleolar localization signal in the N-terminal region of SPIN1 that is essential for its enrichment and function in the nucleolus. We also discovered that, in addition to its H3K4me3 recognizing activity, the H3R8me2a-recognizing capacity of SPIN1 is also indispensable for stimulating rRNA expression. Chromatin immunoprecipitation results indicated that SPIN1 is required for the association or assembly of selective factor 1 (SL1) complex, probably facilitating the initiation of rDNA transcription through its H3 K4me3-R8me2a reader function.