7,345 results on '"RNA Polymerase I"'
Search Results
52. <scp>RNA</scp> polymerase I inhibition induces terminal differentiation, growth arrest, and vulnerability to senolytics in colorectal cancer cells
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Christoph Otto, Carolin Kastner, Stefanie Schmidt, Konstantin Uttinger, Apoorva Baluapuri, Sarah Denk, Mathias T. Rosenfeldt, Andreas Rosenwald, Florian Roehrig, Carsten P. Ade, Christina Schuelein‐Voelk, Markus E. Diefenbacher, Christoph‐Thomas Germer, Elmar Wolf, Martin Eilers, and Armin Wiegering
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Ribosomal Proteins ,Cancer Research ,General Medicine ,Mice ,Oncology ,RNA Polymerase I ,Senotherapeutics ,Genetics ,Animals ,Humans ,Molecular Medicine ,ddc:610 ,Colorectal Neoplasms ,Cell Nucleolus - Abstract
Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside.
- Published
- 2022
53. The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2
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Lucas Tafur, Yashar Sadian, Jonas Hanske, Rene Wetzel, Felix Weis, and Christoph W Müller
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RNA polymerase I ,transcription regulation ,elongation complex ,ribosomal RNA synthesis ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.
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- 2019
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54. TBP facilitates RNA Polymerase I transcription following mitosis.
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Kwan JZJ, Nguyen TF, and Teves SS
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- Animals, Mice, Mouse Embryonic Stem Cells metabolism, Mouse Embryonic Stem Cells cytology, Protein Binding, DNA, Ribosomal genetics, DNA, Ribosomal metabolism, RNA Polymerase I metabolism, RNA Polymerase I genetics, TATA-Box Binding Protein metabolism, TATA-Box Binding Protein genetics, Mitosis, Promoter Regions, Genetic, Transcription, Genetic
- Abstract
The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.
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- 2024
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55. Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.
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Toro-Ascuy D, Cárdenas M, Vásquez-Martínez Y, and Cortez-San Martín M
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- Animals, DNA, Complementary genetics, Cell Line, RNA, Viral genetics, Salmon genetics, Mammals genetics, Isavirus genetics, Orthomyxoviridae genetics, Orthomyxoviridae Infections veterinary, Fish Diseases
- Abstract
The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2024
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56. Reverse Genetics of Hepatitis C Virus Using an RNA Polymerase I-Mediated Transcription.
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Suzuki R and Suzuki T
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- Humans, Hepacivirus genetics, Hepacivirus metabolism, RNA Polymerase I genetics, RNA Polymerase I metabolism, Reverse Genetics, Transfection, DNA, Complementary genetics, RNA, Viral genetics, Hepatitis C genetics, Carcinoma, Hepatocellular genetics
- Abstract
The reverse genetics system commonly used for the production of hepatitis C virus (HCV), which is a major causative agent of liver diseases, involves introduction of the viral genomic RNA synthesized in vitro into human hepatoma cells by electroporation. As an alternative methodology, we describe a cell culture system based on transfection with an expression plasmid containing a full-length HCV cDNA clone flanked by RNA polymerase I promoter and terminator sequences to generate infectious virus particles from transfected cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2024
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57. Inhibition of TAF1B impairs ribosome biosynthesis and suppresses cell proliferation in stomach adenocarcinoma through promoting c-MYC mRNA degradation.
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Chen HF, Li ZP, Wu Q, Yu C, Yan JY, Bai YF, Zhu SM, Qian MX, Liu M, Xu LF, Peng Z, and Zhang F
- Abstract
Hyperactivation of ribosome biosynthesis (RiBi) is a hallmark of cancer, and targeting ribosome biogenesis has emerged as a potential therapeutic strategy. The depletion of TAF1B , a major component of selectivity factor 1 ( SL1 ), disrupts the pre-initiation complex, preventing RNA polymerase I from binding ribosomal DNA and inhibiting the hyperactivation of RiBi. Here, we investigate the role of TAF1B , in regulating RiBi and proliferation in stomach adenocarcinoma (STAD). We disclosed that the overexpression of TAF1B correlates with poor prognosis in STAD, and found that knocking down TAF1B effectively inhibits STAD cell proliferation and survival in vitro and in vivo. TAF1B knockdown may also induce nucleolar stress, and promote c-MYC degradation in STAD cells. Furthermore, we demonstrate that TAF1B depletion impairs rRNA gene transcription and processing, leading to reduced ribosome biogenesis. Collectively, our findings suggest that TAF1B may serve as a potential therapeutic target for STAD and highlight the importance of RiBi in cancer progression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)
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- 2023
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58. Nucleolar structure connects with global nuclear organization.
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Wang C, Ma H, Baserga SJ, Pederson T, and Huang S
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- Humans, HeLa Cells, RNA Polymerase I, Cell Nucleolus, Cell Nucleus
- Abstract
The nucleolus is a multifunctional nuclear body. To tease out the roles of nucleolar structure without resorting to the use of multi-action drugs, we knocked down the RNA polymerase I subunit RPA194 in HeLa cells by siRNA. Loss of RPA194 resulted in nucleolar-structural segregation and effects on both nucleolus-proximal and distal-nuclear components. The perinucleolar compartment was disrupted, centromere clustering around nucleoli was significantly reduced, and the intranuclear locations of specific genomic loci were altered. Moreover, Cajal bodies, distal from nucleoli, underwent morphological and some compositional changes. In comparison, when the preribosomal RNA-processing factor, UTP4, was knocked down, neither nucleolar segregation nor the intranuclear effects were observed, demonstrating that the changes of nucleolar proximal and distal nuclear domains in RPA194 knockdown cells unlikely arise from a cessation of ribosome synthesis, rather from the consequence of nucleolar-structure alteration. These findings point to a commutative system that links nucleolar structure to the maintenance and spatial organization of certain nuclear domains and genomic loci.
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- 2023
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59. Beyond rRNA: nucleolar transcription generates a complex network of RNAs with multiple roles in maintaining cellular homeostasis
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Shuang Feng and James L. Manley
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Transcription, Genetic ,Macromolecular Substances ,RNA Polymerase I ,RNA, Ribosomal ,Genetics ,RNA Precursors ,Homeostasis ,DNA, Intergenic ,RNA Polymerase II ,DNA, Ribosomal ,Cell Nucleolus ,Developmental Biology - Abstract
Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes ∼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.
- Published
- 2023
60. Coordinated Control of rRNA Processing by RNA Polymerase I.
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Scull, Catherine E. and Schneider, David A.
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RIBOSOMAL RNA , *RNA polymerases , *TRANSCRIPTION factors , *CELL growth , *TECHNOLOGICAL innovations , *RIBOSOMES - Abstract
Ribosomal RNA (rRNA) is co- and post-transcriptionally processed into active ribosomes. This process is dynamically regulated by direct covalent modifications of the polymerase that synthesizes the rRNA, RNA polymerase I (Pol I), and by interactions with cofactors that influence initiation, elongation, and termination activities of Pol I. The rate of transcription elongation by Pol I directly influences processing of nascent rRNA, and changes in Pol I transcription rate result in alternative rRNA processing events that lead to cellular signaling alterations and stress. It is clear that in divergent species, there exists robust organization of nascent rRNA processing events during transcription elongation. This review evaluates the current state of our understanding of the complex relationship between transcription elongation and rRNA processing. Transcription elongation rate by RNA polymerase I dynamically controls ribosome biogenesis directly. Conservation of transcription factors and processing pathways suggests that dynamic regulation of Pol I is conserved from lower to higher eukaryotes. Dysregulation of transcription by Pol I and pre-rRNA processing is observed in several diseases, resulting in altered cell growth and signaling pathways. Emerging technologies both in vivo and in vitro will allow for a better understanding of the dynamic nature of ribosome biogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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61. Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei.
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Budzak, James, Kerry, Louise E., Aristodemou, Aris, Hall, Belinda S., Witmer, Kathrin, Kushwaha, Manish, Davies, Carys, Povelones, Megan L., McDonald, Jacquelyn R., Sur, Aakash, Myler, Peter J., and Rudenko, Gloria
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TRYPANOSOMA brucei , *RNA polymerases , *ANTIGENIC variation , *DNA - Abstract
Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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62. Association of TATA box-binding protein-associated factor RNA polymerase I subunit C (TAF1C) with T2DM.
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Abdulwahab, Rabab Asghar, Allaith, Abdul Ameer A., Shinwari, Zakia, Alaiya, Ayodele, and Giha, Hayder A.
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RNA polymerase I , *TYPE 2 diabetes , *BIOLOGICAL tags , *MASS spectrometry , *LIQUID chromatography - Abstract
Proteins differential expression in type 2 diabetes mellitus (T2DM) can be due to etiological factors or pathological changes, such proteins can be utilized as biomarkers. Identification of a marker protein out of thousands became a feasible task during the proteomics era by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, blood samples were obtained from 80 Bahraini subjects with and without T2DM, a subset was used for proteomic analysis by LC-MS/MS, while all samples were used for ELISA analysis of 3 proteins, TATA-box binding protein-associated factor RNA polymerase-1-C (TAF1C), ceruloplasmin (CERP) and fibronectin (FN). The former 2 proteins were selected from the T2DM-protein-panel identified by LC-MS/MS, and the latter was analyzed for validation of the setting. The main findings of the proteomic analysis are i. Identifications of 62 differentially expressed proteins in T2DM, ii. Upregulation of 71% of the identified proteins. While the ELISA analysis showed that; both TAF1C and FN were significantly increased in T2DM (P 0.015 and P 0.001, respectively), while CERP was not (P 0.088). Logistic regression analysis: i. confirmed the above associations after correction for covariates, ii. Revealed an interaction between age and gender that affect the association of the proteins with T2DM. In conclusion, knowing that TAF1C is a prerequisite in ribosomal biogenesis, our ELISA results are suggestive of increased protein synthesis in T2DM, explaining the observed upregulation of the proteins identified by LC-MSMS. The association between T2DM and TAF1C is a novel finding that might open a new avenue in DM research. • LC-MS/MS proteomic analysis revealed novel association of 37 peptides (including TAF1C & CERP) with T2DM. • ELISA confirmed the strong association of TAF1C and weak association of CERP peptides with T2DM. • Novel association of TAF1C with T2DM suggests increased ribosomal biogenesis. • ELISA disclosed marked elevation of blood fibronectin (Fn) inT2DM, and validated the setting. • Age/gender interaction affects the association of the above peptides with T2DM. [ABSTRACT FROM AUTHOR]
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- 2019
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63. Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells.
- Author
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Lei, Hu, Tu, Yaoyao, Yang, Li, Jin, Jin, Luo, Hao, Xu, Hanzhang, Kang, Jingwu, Zhou, Li, and Wu, Yingli
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ANTIMALARIALS , *APOPTOSIS , *CELL lines , *LYMPHOBLASTIC leukemia , *MICE , *PROTEIN-tyrosine kinases , *RNA , *STEM cells , *TRANSFERASES , *CHRONIC myeloid leukemia , *IN vitro studies , *IN vivo studies , *PHARMACODYNAMICS - Abstract
Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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64. Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.
- Author
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Darrière, Tommy, Pilsl, Michael, Sarthou, Marie-Kerguelen, Chauvier, Adrien, Genty, Titouan, Audibert, Sylvain, Dez, Christophe, Léger-Silvestre, Isabelle, Normand, Christophe, Henras, Anthony K., Kwapisz, Marta, Calvo, Olga, Fernández-Tornero, Carlos, Tschochner, Herbert, and Gadal, Olivier
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RNA polymerase I , *POINT mutation (Biology) , *DNA-binding proteins , *SUPPRESSOR mutation , *RIBOSOMAL RNA - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2019
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65. Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.
- Author
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Michael Babokhov, Tadasu Nozaki, Sachiko Tamura, Ryosuke Nagashima, Kayo Hibino, Ryosuke Imai, Kazuhiro Maeshima, Kanemaki, Masato T., Ashwin, S. S., Shin Fujishiro, Masaki Sasai, Tomomi Tani, Michael Shribak, and Hiroshi Kimura
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GENOMES , *CHROMATIN , *RNA polymerase I - Abstract
Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets. [ABSTRACT FROM AUTHOR]
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- 2019
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66. In yeast cells arrested at the early S-phase by hydroxyurea, rRNA gene promoters and chromatin are poised for transcription while rRNA synthesis is compromised.
- Author
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Charton, Romain, Muguet, Alexia, Griesenbeck, Joachim, Smerdon, Michael J., and Conconi, Antonio
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RIBONUCLEOSIDE diphosphate reductase , *RIBOSOMAL RNA , *CHROMATIN , *RNA polymerases , *DNA replication , *GENETIC regulation - Abstract
• In early S-phase arrested cells by HU essentially all rRNA genes have an open chromatin. • Only a portion of all open rRNA genes has RNA polymerases I. • rRNA genes remain poised for transcription in cell cycle arrested cells. • rRNA processing and synthesis are compromised in HU treated cells. Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase that is used as a chemotherapeutic agent to treat a number of chronic diseases. Addition of HU to cell cultures causes reduction of the dNTP cellular pool below levels that are required for DNA replication. This trigger dividing cells to arrest in early S-phase of the cell cycle. Cell division hinges on ribosome biogenesis, which is tightly regulated by rRNA synthesis. Remarkably, HU represses the expression of some genes the products of which are required for rRNA maturation. To gain more information on the cellular response to HU, we employed the yeast Saccharomyces cerevisiae as model organism and analyzed the changing aspects of closed to open forms of rRNA gene chromatin during cell cycle arrest, the arrangement of RNA polymerase-I (RNAPI) on the open genes, the presence of RNAPI transcription-factors, transcription and rRNA maturation. The rRNA gene chromatin structure was analyzed by psoralen crosslinking and the distribution of RNAPI was investigated by chromatin endogenous cleavage. In HU arrested cells nearly all rRNA genes were in the open form of chromatin, but only a portion of them was engaged with RNAPI. Analyses by chromatin immuno-precipitation confirmed that the overall formation of transcription pre-initiation complexes remained unchanged, suggesting that the onset of rRNA gene activation was not significantly affected by HU. Moreover, the in vitro transcription run-on assay indicated that RNAPI retained most of its transcription elongation activity. However, in HU treated cells, we found that: (1) RNAPI accumulated next to the 5′-end of rRNA genes; (2) considerably less rRNA filaments were observed in electron micrographs of rDNA transcription units; and (3) rRNA maturation was compromised. It is established that HU inhibition of ribonucleotide reductase holds back DNA replication. This study indicates a hitherto unexplored cellular response to HU, namely altered rRNA synthesis, which could participate to hamper cell division. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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67. Nuclear Phosphatidylinositol 5-Phosphatase Is Essential for Allelic Exclusion of Variant Surface Glycoprotein Genes in Trypanosomes.
- Author
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Cestari, Igor, McLeland-Wieser, Hilary, and Stuart, Kenneth
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GLYCOPROTEIN genetics , *PHOSPHOINOSITIDES , *TELOMERES , *DEPHOSPHORYLATION , *TRANSCRIPTION factor AP-1 - Abstract
Allelic exclusion of variant surface glycoprotein (VSG) genes is essential for African trypanosomes to evade the host antibody response by antigenic variation. The mechanisms by which this parasite expresses only one of its ~2,000 VSG genes at a time are unknown. We show that nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) interacts with repressor activator protein 1 (RAP1) in a multiprotein complex and functions in the control of VSG allelic exclusion. RAP1 binds PIP5Pase substrate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], and catalytic mutation of PIP5Pase that inhibits PI(3,4,5)P3 dephosphorylation results in simultaneous transcription of VSGs from all telomeric expression sites (ESs) and from silent subtelomeric VSG arrays. PIP5Pase and RAP1 bind to telomeric ESs, especially at 70-bp repeats and telomeres, and their binding is altered by PIP5Pase inactivation or knockdown, implying changes in ES chromatin organization. Our data suggest a model whereby PIP5Pase controls PI(3,4,5)P3 binding by RAP1 and, thus, RAP1 silencing of telomeric and subtelomeric VSG genes. Hence, allelic exclusion of VSG genes may entail control of nuclear phosphoinositides. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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68. Differential Symptom Development and Viral RNA Loads in 10 Nicotiana benthamiana Accessions Infected with the Tobamovirus Yellow Tailflower Mild Mottle Virus
- Author
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Xin Chen, Yuxia Guo, Michael G. K. Jones, Krishnapillai Sivasithamparam, Hua Li, Stephen J. Wylie, and Weinan Xu
- Subjects
Genetic diversity ,biology ,Inoculation ,viruses ,fungi ,Mutant ,food and beverages ,Nicotiana benthamiana ,Tobamovirus ,Plant Science ,biology.organism_classification ,Virology ,Virus ,RNA polymerase I ,Agronomy and Crop Science ,Gene - Abstract
Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 (the lab type), which has a mutant, functionally defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done regarding responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harbored wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days postinoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried more than twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, in which viral RNA load was low until 15 dpi, after which it increased more than 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the 10 N. benthamiana accessions in some cases reflected geographical location, but in other accessions this was not so.
- Published
- 2022
69. NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I.
- Author
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Clarke, Andrew M., Engel, Krysta L., Giles, Keith E., Petit, Chad M., and Schneider, David A.
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NUCLEOTIDE sequencing , *RNA polymerase I , *GENETIC transcription , *NUCLEOTIDES , *RIBOSOMAL DNA , *PROTEIN elongation factor genetics , *GENETIC regulation - Abstract
DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae. We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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70. Polymerase-1 pathway activation in acute multiple sclerosis relapse.
- Author
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Achiron, Anat, Zilkha-Falb, Rina, Feldman, Anna, Bovim, Maria, Rosenblum, Onn, Sarova-Pinhas, Ida, Magalashvili, David, Dolev, Mark, Menascu, Shay, and Gurevich, Michael
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MULTIPLE sclerosis , *RNA polymerase I , *GENE expression , *DISEASE relapse , *OPTIC neuritis - Abstract
Abstract Background Increased expression of RNA polymerase 1 (POL1) molecular pathway was reported to be associated with increased disease activity in patients with multiple sclerosis (MS). However, the operating molecular mechanisms that characterize the pattern of acute MS relapse activity has not been thoroughly studied. Objective To assess POL1 pathway expression during acute MS relapse. Methods We studied POL1 pathway associated biomarkers during the first acute optic neuritis attack of MS, and in relapsing-remitting MS patients treated with disease-modifying drugs (DMDs) experiencing an acute MS relapse or a radiological relapse using gene expression microarrays and quantitative RT-PCR. Results In MS patients (N = 6) during the first acute optic neuritis attack POL1 pathway activation was evident by over-expression of POL1 related network including transcription factor UBTF and downstream components of Assembly of RNA POL1 complex (p=1.92E-03). POL1 related biomarkers RRN3, POLR1D and LRPPRC were over-expressed x1.6 (p =.002), ×1.7 (p =.01) and x2.0 (p =.001) times higher respectively, in MS patients (N = 30) during acute clinical relapse as compared with remission. Similarly, in MS patients (N = 21) that presented with a radiological relapse, we observed significant activation of POL1 related biomarkers including RRN3 (p =.01), POLR1D (p =.002), POLR1E (p =.0001) and LRPPRC (p =.006), as compared with remission, as well as overexpression of a large group of genes encoding ribosomal proteins like RPS6KA3 (p = 7.2E-6), RRP8 (p =.0002) and RPCS9 (p =.0008). Conclusions Our findings demonstrated increased POL1 pathway activity in acute MS relapse and suggest that targeted inactivation of POL1 pathway represent a novel strategy for a better treatment of acute MS relapse. [ABSTRACT FROM AUTHOR]
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- 2018
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71. Temperature shift activates bloodstream VSG expression site promoters in Trypanosoma brucei.
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Kolev, Nikolay G., Ramsdell, Trisha K., and Tschudi, Christian
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GLYCOPROTEINS , *GENE expression , *TRYPANOSOMA brucei , *BODY temperature , *RNA polymerase I - Abstract
Graphical abstract Highlights • Metacyclic VSG expression site (ES) promoters are insensitive to changes in temperature. • Bloodstream VSG ES (BES) promoters are repressed at ambient temperature. • BES promoters are activated at mammalian body temperature. Abstract Trypanosoma brucei relies on two types of variant surface glycoprotein (VSG) expression sites (ESs) for RNA polymerase I (Pol I) transcription of VSG pre-mRNA. Trypanosomes developing into infectious metacyclic cells in the tsetse vector use metacyclic VSG ESs (MESs) and proliferating parasites in the mammalian host deploy bloodstream VSG ESs (BESs). Unlike the monocistronic MESs, BESs are polycistronic and their highly conserved promoters differ considerably from the MES promoters. The significance of the divergent sequences of MES and BES promoters remains to be determined. We used a reporter system to specifically test the effect of temperature on the activity of MES and BES promoters in procyclic trypanosomes and our results demonstrate that transcription from the MES promoter is largely insensitive to changes in temperature. In contrast, the BES promoter drives rapid activation of transcription upon a change of temperature from 28 °C to 37 °C. Additionally, endogenous BESs respond similarly to the elevation of temperature and initiate increased production of BES pre-mRNA and mRNA. Our results indicate that the sequence of the BES promoter is a specificity signal which triggers BES activation in vivo upon entry into the mammalian host. [ABSTRACT FROM AUTHOR]
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- 2018
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72. Processing and roles of snoRNA-ended long noncoding RNAs.
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Xing, Yu-Hang and Chen, Ling-Ling
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- *
NON-coding RNA , *CELL proliferation , *RIBOSOMAL RNA , *ORIGIN of life , *MOLECULAR genetics - Abstract
Small nucleolar RNAs (snoRNAs) are a family of conserved nuclear RNAs that function in the modification of small nuclear RNAs (snRNAs) or ribosomal RNAs (rRNAs), or participate in the processing of rRNAs during ribosome subunit maturation. Eukaryotic DNA transcription and RNA processing produce many long noncoding RNA (lncRNA) species. Although most lncRNAs are processed like typical mRNAs to be 5' capped and 3' polyadenylated, other types of lncRNAs are stabilized from primary Pol II transcripts by alternative mechanisms. One way to generate stable lncRNAs is to co-operate with snoRNA processing to produce snoRNA-ended lncRNAs (sno-lncRNAs) and 5' snoRNA-ended and 3'-polyadenylated lncRNAs (SPAs). Rather than silently accumulating in the nucleus, some sno-lncRNAs and SPAs are involved in the regulation of pre-rRNA transcription and alternative splicing of pre-mRNAs. Here we provide a mini-review to discuss the biogenesis and functions of these unusually processed lncRNAs. [ABSTRACT FROM AUTHOR]
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- 2018
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73. The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.
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Fuller, Kaila B., Jacobs, Ruth Q., Schneider, David A., and Lucius, Aaron L.
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RIBOSOMAL RNA , *EXONUCLEASES , *RNA polymerases , *BIOSYNTHESIS - Abstract
[Display omitted] • ΔA12 Pol I exhibits reversible kinetics when catalyzing nucleotide addition. • The A12.2 subunit promotes fast PPi release. • WT and ΔA12 Pol I rate-limiting step in nucleotide addition is bond formation. RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity. [ABSTRACT FROM AUTHOR]
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- 2023
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74. An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes
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James Budzak, Robert Jones, Christian Tschudi, Nikolay G. Kolev, Gloria Rudenko, and Wellcome Trust
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Membrane Glycoproteins ,Multidisciplinary ,Organisms, Genetically Modified ,Transcription, Genetic ,RNA Splicing ,Science ,Trypanosoma brucei brucei ,Protozoan Proteins ,General Physics and Astronomy ,General Chemistry ,Microbiology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Parasite biology ,Gene Expression Regulation ,RNA Polymerase I ,Nuclear Bodies ,parasitic diseases ,RNA, Messenger ,Cells, Cultured ,Variant Surface Glycoproteins, Trypanosoma - Abstract
A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated., A Variant Surface Glycoprotein (VSG) coat protects bloodstream form T. brucei. Applying super-resolution microscopy Budzak et al. characterize a set of nuclear bodies, which associate with the active expression site in bloodstream form T. brucei and highlight the importance of trans-splicing for transcription of VSG.
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- 2022
75. Dynamic regulation and requirement for ribosomal RNA transcription during mammalian development
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Karla T. Falcon, Kristin E. N. Watt, Soma Dash, Ruonan Zhao, Daisuke Sakai, Emma L. Moore, Sharien Fitriasari, Melissa Childers, Mihaela E. Sardiu, Selene Swanson, Dai Tsuchiya, Jay Unruh, George Bugarinovic, Lin Li, Rita Shiang, Annita Achilleos, Jill Dixon, Michael J. Dixon, and Paul A. Trainor
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Craniofacial Abnormalities ,Ribosomal Proteins ,Mice ,Multidisciplinary ,Transcription, Genetic ,Neural Crest ,RNA Polymerase I ,RNA, Ribosomal ,Skull ,Animals ,Proto-Oncogene Proteins c-mdm2 ,Tumor Suppressor Protein p53 ,Mandibulofacial Dysostosis - Abstract
Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunitsPolr1a,Polr1c, and associated factorTcof1in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis–Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.
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- 2023
76. Conserved Curvature of RNA Polymerase I Core Promoter Beyond rRNA Genes: The Case of the Tritryps
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Pablo Smircich, María Ana Duhagon, and Beatriz Garat
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Intrinsic curvature ,RNA polymerase I ,Core promoter ,Trypanosoma ,Leishmania ,Biology (General) ,QH301-705.5 ,Computer applications to medicine. Medical informatics ,R858-859.7 - Abstract
In trypanosomatids, the RNA polymerase I (RNAPI)-dependent promoters controlling the ribosomal RNA (rRNA) genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no conformational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such conserved curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.
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- 2015
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77. Nucleolar GTPase Bms1 displaces Ttf1 from RFB-sites to balance progression of rDNA transcription and replication
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Jian Hong, Jun Chen, Qinfang Zhu, Yinan He, Hong Chen, Yong Wang, Yanqing Zhu, Jinrong Peng, Jun Huang, Yunqin Li, Ling Huang, Li Jan Lo, Jinhua Han, and Boxiang Tao
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DNA Replication ,Nucleolus ,GTPase ,AcademicSubjects/SCI01180 ,Ttf1 ,DNA, Ribosomal ,GTP Phosphohydrolases ,Transcription (biology) ,RNA Polymerase I ,Genetics ,RNA polymerase I ,Animals ,nucleolus ,Molecular Biology ,biology ,Bms1 ,RNA ,replication-fork barrier ,Cell Biology ,General Medicine ,Articles ,Ribosomal RNA ,Cell cycle ,zebrafish ,Proliferating cell nuclear antigen ,Cell biology ,Editor's Choice ,ribosome small subunit processome ,RNA, Ribosomal ,biology.protein ,cell cycle - Abstract
18S, 5.8S, and 28S ribosomal RNAs (rRNAs) are cotranscribed as a pre-ribosomal RNA (pre-rRNA) from the rDNA by RNA polymerase I whose activity is vigorous during the S-phase, leading to a conflict with rDNA replication. This conflict is resolved partly by replication-fork-barrier (RFB)-sites sequences located downstream of the rDNA and RFB-binding proteins such as Ttf1. However, how Ttf1 is displaced from RFB-sites to allow replication fork progression remains elusive. Here, we reported that loss-of-function of Bms1l, a nucleolar GTPase, upregulates rDNA transcription, causes replication-fork stall, and arrests cell cycle at the S-to-G2 transition; however, the G1-to-S transition is constitutively active characterized by persisting DNA synthesis. Concomitantly, ubf, tif-IA, and taf1b marking rDNA transcription, Chk2, Rad51, and p53 marking DNA-damage response, and Rpa2, PCNA, Fen1, and Ttf1 marking replication fork stall are all highly elevated in bms1l mutants. We found that Bms1 interacts with Ttf1 in addition to Rc1l. Finally, we identified RFB-sites for zebrafish Ttf1 through chromatin immunoprecipitation sequencing and showed that Bms1 disassociates the Ttf1‒RFB complex with its GTPase activity. We propose that Bms1 functions to balance rDNA transcription and replication at the S-phase through interaction with Rcl1 and Ttf1, respectively. TTF1 and Bms1 together might impose an S-phase checkpoint at the rDNA loci.
- Published
- 2021
78. RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21
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Ruth Q. Jacobs, Kaila B. Fuller, Stephanie L. Cooper, Zachariah I. Carter, Marikki Laiho, Aaron L. Lucius, and David A. Schneider
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Cancer Research ,Oncology ,RNA polymerase I ,RNA polymerase II ,RNA polymerase III ,BMH-21 ,cancer therapeutics ,transcription elongation - Abstract
Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.
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- 2022
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79. Hammerhead Ribozymes Against Virus and Viroid RNAs
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Carbonell, Alberto, Flores, Ricardo, Gago, Selma, Erdmann, Volker A., editor, and Barciszewski, Jan, editor
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- 2012
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80. Autophagy deficiency activates rDNA transcription
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Wei Wan, Yinfeng Xu, Qian Wang, Lijiang Song, Hui Zhang, Zhuo Ren, Yaosen Wu, Zhengfu He, Chuying Qian, and Lei Wang
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RNA polymerase II ,P70-S6 Kinase 1 ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,DNA, Ribosomal ,Sequestosome 1 ,RNA Polymerase I ,Transcription (biology) ,Sequestosome-1 Protein ,Autophagy ,RNA polymerase I ,education ,Molecular Biology ,Transcription factor ,Mechanistic target of rapamycin ,Sirolimus ,education.field_of_study ,Cell Biology ,Cell biology ,RNA, Ribosomal ,Ribosomal protein s6 ,biology.protein ,RNA ,Apoptosis Regulatory Proteins ,Carrier Proteins ,Research Paper - Abstract
Macroautophagy/autophagy, a highly conserved lysosome-dependent degradation pathway, has been intensively studied in regulating cell metabolism by degradation of intracellular components. In this study, we link autophagy to RNA metabolism by uncovering a regulatory role of autophagy in ribosomal RNA (rRNA) synthesis. Autophagy-deficient cells exhibit much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling and promotes the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoters, which leads to an increase of 47S rRNA transcribed from rDNA. Functionally, autophagy deficiency promotes protein synthesis, cell growth and cell proliferation, both of which are dependent on SQSTM1 accumulation. Taken together, our findings suggest that autophagy deficiency is involved in RNA metabolism by activating rDNA transcription and provide novel mechanisms for the reprogramming of cell metabolism in autophagy-related diseases including multiple types of cancers. Abbreviations: 5-FUrd: 5-fluorouridine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A/RPA194: RNA polymerase I subunit A; POLR2A: RNA polymerase II subunit A; rDNA: ribosomal DNA; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; rRNA: ribosomal RNA; RUBCN/Rubicon: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; SUnSET: surface sensing of translation; TAX1BP1: Tax1 binding protein 1; UBTF/UBF1: upstream binding transcription factor; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild-type.
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- 2021
81. Nucleolar TFIIE plays a role in ribosomal biogenesis and performance
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Tamara Phan, Pallab Maity, Christina Ludwig, Lisa Streit, Miltiadis Tsesmelis, Sebastian Iben, Karin Scharffetter-Kochanek, and Jens Michaelis
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Proteasome Endopeptidase Complex ,Hot Temperature ,Transcription, Genetic ,AcademicSubjects/SCI00010 ,Ribosome biogenesis ,RNA polymerase II ,Biology ,Ribosome ,Transcription Factors, TFII ,Transcription (biology) ,Genes, Reporter ,RNA Polymerase I ,Genetics ,RNA polymerase I ,Humans ,Trichothiodystrophy Syndromes ,Luciferases ,Molecular Biology ,Cell Line, Transformed ,Organelle Biogenesis ,General transcription factor ,Protein Stability ,RNA ,Ribosomal RNA ,Fibroblasts ,Cell biology ,Gene Expression Regulation ,RNA, Ribosomal ,Protein Biosynthesis ,Mutation ,biology.protein ,Proteostasis ,Ribosomes ,Transcription Factor TFIIH ,Cell Nucleolus - Abstract
Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here, we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE—due to impaired ribosomal biogenesis and translational accuracy—lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.
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- 2021
82. Structure of an inactive RNA polymerase II dimer
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Patrick Cramer, Christian Dienemann, and Shintaro Aibara
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Models, Molecular ,biology ,AcademicSubjects/SCI00010 ,DNA polymerase II ,viruses ,Cryoelectron Microscopy ,Sus scrofa ,RNA ,RNA polymerase II ,Saccharomyces cerevisiae ,RNA polymerase III ,Biochemistry ,Transcription (biology) ,Structural Biology ,Genetics ,biology.protein ,RNA polymerase I ,Animals ,RNA Polymerase II ,DNA polymerase I ,Protein Multimerization ,Dimerization ,Polymerase - Abstract
Eukaryotic gene transcription is carried out by three RNA polymerases: Pol I, Pol II and Pol III. Although it has long been known that Pol I can form homodimers, it is unclear whether and how the two other RNA polymerases dimerize. Here we present the cryo-electron microscopy (cryo-EM) structure of a mammalian Pol II dimer at 3.5 Å resolution. The structure differs from the Pol I dimer and reveals that one Pol II copy uses its RPB4-RPB7 stalk to penetrate the active centre cleft of the other copy, and vice versa, giving rise to a molecular handshake. The polymerase clamp domain is displaced and mobile, and the RPB7 oligonucleotide-binding fold mimics the DNA–RNA hybrid that occupies the cleft during active transcription. The Pol II dimer is incompatible with nucleic acid binding as required for transcription and may represent an inactive storage form of the polymerase.
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- 2021
83. Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans
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Xiangyang Chen, Shimiao Liao, Chengming Zhu, Ting Xu, Zongxiu Xu, Demin Xu, Xufei Zhou, Qile Jin, Shouhong Guang, and Xuezhu Feng
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Small interfering RNA ,Transcription Elongation, Genetic ,Exosome complex ,Nucleolus ,AcademicSubjects/SCI00010 ,genetic processes ,Gene regulation, Chromatin and Epigenetics ,RNA ,DNA-Directed RNA Polymerases ,Biology ,Ribosomal RNA ,Exosomes ,Cell biology ,RNA interference ,Transcription (biology) ,RNA, Ribosomal ,Mutation ,Genetics ,RNA polymerase I ,Animals ,Gene Silencing ,RNA, Small Interfering ,Caenorhabditis elegans ,Cell Nucleolus - Abstract
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.
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- 2021
84. HSF1 induces RNA polymerase II synthesis of ribosomal RNA in S. cerevisiae during nitrogen deprivation
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Merita Kabashi, Matt Haymowicz, Kushal Bhatt, Arjuna Rao Vallabhaneni, Violet Wayman, Shazia A. Ahmed, and Heather Conrad-Webb
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Chromatin Immunoprecipitation ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Nitrogen ,RNA polymerase II ,Polymerase switch ,Saccharomyces cerevisiae ,Hsf1 ,Ribosome ,Models, Biological ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Genetics ,RNA polymerase I ,Promoter Regions, Genetic ,Transcription factor ,Polymerase ,Heat-Shock Proteins ,biology ,RNA ,General Medicine ,Ribosomal RNA ,Cell biology ,DNA-Binding Proteins ,rRNA synthesis ,Genetic Loci ,RNA, Ribosomal ,biology.protein ,Original Article ,RNA Polymerase II ,Nitrogen deprivation ,Transcription Factors - Abstract
The resource intensive process of accurate ribosome synthesis is essential for cell viability in all organisms. Ribosome synthesis regulation centers on RNA polymerase I (pol I) transcription of a 35S rRNA precursor that is processed into the mature 18S, 5.8S and 25S rRNAs. During nutrient deprivation or stress, pol I synthesis of rRNA is dramatically reduced. Conversely, chronic stress such as mitochondrial dysfunction induces RNA polymerase II (pol II) to transcribe functional rRNA using an evolutionarily conserved cryptic pol II rDNA promoter suggesting a universal phenomenon. However, this polymerase switches and its role in regulation of rRNA synthesis remain unclear. In this paper, we demonstrate that extended nitrogen deprivation induces the polymerase switch via components of the environmental stress response. We further show that the switch is repressed by Sch9 and activated by the stress kinase Rim15. Like stress-induced genes, the switch requires not only pol II transcription machinery, including the mediator, but also requires the HDAC, Rpd3 and stress transcription factor Hsf1. The current work shows that the constitutive allele, Hsf1PO4* displays elevated levels of induction in non-stress conditions while binding to a conserved site in the pol II rDNA promoter upstream of the pol I promoter. Whether the polymerase switch serves to provide rRNA when pol I transcription is inhibited or fine-tunes pol I initiation via RNA interactions is yet to be determined. Identifying the underlying mechanism for this evolutionary conserved phenomenon will help understand the mechanism of pol II rRNA synthesis and its role in stress adaptation.
- Published
- 2021
85. TAF1B depletion leads to apoptotic cell death by inducing nucleolar stress and activating p53-miR-101 circuit in hepatocellular carcinoma.
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Chen HF, Gao DD, Jiang XQ, Sheng H, Wu Q, Zheng Q, Zhai QC, Yuan L, Liu M, Xu LF, Qian MX, Xu H, Fang J, and Zhang F
- Abstract
Background: TAF1B (TATA Box Binding Protein (TBP)-Associated Factor) is an RNA polymerase regulating rDNA activity, stress response, and cell cycle. However, the function of TAF1B in the progression of hepatocellular carcinoma (HCC) is unknown., Objective: In this study, we intended to characterize the crucial role and molecular mechanisms of TAF1B in modulating nucleolar stress in HCC., Methods: We analyzed the differential expression and prognostic value of TAF1B in hepatocellular carcinoma based on The Cancer Genome Atlas (TCGA) database, tumor and paraneoplastic tissue samples from clinical hepatocellular carcinoma patients, and typical hepatocellular carcinoma. We detected cell proliferation and apoptosis by lentiviral knockdown of TAF1B expression levels in HepG2 and SMMC-7721 cells using clone formation, apoptosis, and Western blotting (WB) detection of apoptosis marker proteins. Simultaneously, we investigated the influence of TAF1B knockdown on the function of the pre-initiation complex (PIC) by WB, and co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays verified the interaction between the complexes and the effect on rDNA activity. Immunofluorescence assays measured the expression of marker proteins of nucleolus stress, fluorescence in situ hybridization (FISH) assays checked the rDNA activity, and qRT-PCR assays tested the pre-rRNA levels. Regarding molecular mechanisms, we investigated the role of p53 and miR-101 in modulating nucleolar stress and apoptosis. Finally, the impact of TAF1B knockdown on tumor growth, apoptosis, and p53 expression was observed in xenograft tumors., Result: We identified that TAF1B was highly expressed in hepatocellular carcinoma and associated with poor prognosis in HCC patients. TAF1B depletion modulated nucleolar stress and apoptosis in hepatocellular carcinoma cells through positive and negative feedback from p53-miR-101. RNA polymerase I transcription repression triggered post-transcriptional activation of miR-101 in a p53-dependent manner. In turn, miR-101 negatively feeds back through direct inhibition of the p53-mediated PARP pathway., Conclusion: These findings broaden our comprehension of the function of TAF1B-mediated nucleolar stress in hepatocellular carcinoma and may offer new biomarkers for exploring prospective therapeutic targets in HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Gao, Jiang, Sheng, Wu, Zheng, Zhai, Yuan, Liu, Xu, Qian, Xu, Fang and Zhang.)
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- 2023
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86. Targeting mutant dicer tumorigenesis in pleuropulmonary blastoma via inhibition of RNA polymerase I.
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Wong MRE, Lim KH, Hee EXY, Chen H, Kuick CH, Aw SJ, Chang KTE, Syed Sulaiman N, Low SY, Hartono S, Tran ANT, Ahamed SH, Lam CMJ, Soh SY, Hannan KM, Hannan RD, Coupland LA, and Loh AHP
- Subjects
- Humans, Tumor Suppressor Protein p53 genetics, Carcinogenesis, Ribonuclease III genetics, Ribonuclease III metabolism, DEAD-box RNA Helicases genetics, DEAD-box RNA Helicases metabolism, RNA Polymerase I genetics, RNA Polymerase I metabolism, Pulmonary Blastoma genetics, Pulmonary Blastoma metabolism, Pulmonary Blastoma pathology
- Abstract
DICER1 mutations predispose to increased risk for various cancers, particularly pleuropulmonary blastoma (PPB), the commonest lung malignancy of childhood. There is a paucity of directly actionable molecular targets as these tumors are driven by loss-of-function mutations of DICER1. Therapeutic development for PPB is further limited by a lack of biologically and physiologically-representative disease models. Given recent evidence of Dicer's role as a haploinsufficient tumor suppressor regulating RNA polymerase I (Pol I), Pol I inhibition could abrogate mutant Dicer-mediated accumulation of stalled polymerases to trigger apoptosis. Hence, we developed a novel subpleural orthotopic PPB patient-derived xenograft (PDX) model that retained both RNase IIIa and IIIb hotspot mutations and recapitulated the cardiorespiratory physiology of intra-thoracic disease, and with it evaluated the tolerability and efficacy of first-in-class Pol I inhibitor CX-5461. In PDX tumors, CX-5461 significantly reduced H3K9 di-methylation and increased nuclear p53 expression, within 24 hours' exposure. Following treatment at the maximum tolerated dosing regimen (12 doses, 30 mg/kg), tumors were smaller and less hemorrhagic than controls, with significantly decreased cellular proliferation, and increased apoptosis. As demonstrated in a novel intrathoracic tumor model of PPB, Pol I inhibition with CX-5461 could be a tolerable and clinically-feasible therapeutic strategy for mutant Dicer tumors, inducing antitumor effects by decreasing H3K9 methylation and enhancing p53-mediated apoptosis., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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87. CMTM6 attenuates cisplatin-induced cell death in OSCC by regulating AKT/c-Myc-driven ribosome biogenesis
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Pallavi Mohapatra, Sibasish Mohanty, Shamima Azma Ansari, Omprakash Shriwas, Arup Ghosh, Rachna Rath, Saroj Kumar Das Majumdar, Rajeeb K. Swain, Sunil K. Raghav, and Rupesh Dash
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Cell Death ,Squamous Cell Carcinoma of Head and Neck ,Mice, Nude ,Mechanistic Target of Rapamycin Complex 1 ,Ligands ,Biochemistry ,DNA, Ribosomal ,B7-H1 Antigen ,Mice ,Head and Neck Neoplasms ,RNA Polymerase I ,Cell Line, Tumor ,Genetics ,Carcinoma, Squamous Cell ,Animals ,Humans ,Mouth Neoplasms ,Cisplatin ,RNA, Small Interfering ,Molecular Biology ,Proto-Oncogene Proteins c-akt ,Ribosomes ,Zebrafish ,Biotechnology - Abstract
CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism of how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin-resistant OSCC lines stably expressing Nt shRNA and CMTM6 shRNA revealed that CMTM6 might be a potential regulator of the ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over-expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 was also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin-resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or pharmacological inhibitor (CX-5461) significantly restores cisplatin-mediated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of the ribosome biogenesis network and targeting the ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.
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- 2022
88. The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans
- Author
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Daiß, Julia L., Pilsl, Michael, Straub, Kristina, Bleckmann, Andrea, Höcherl, Mona, Heiss, Florian B., Abascal-Palacios, Guillermo, Ramsay, Ewan Phillip, Tlučková, Katarina, Mars, Jean-Clement, Fürtges, Torben, Bruckmann, Astrid, Rudack, Till, Bernecky, Carrie, Lamour, Valérie, Panov, Konstantin, Vannini, Alessandro, Moss, Tom, Engel, Christoph, Universität Regensburg (UR), Universidad del Pais Vasco / Euskal Herriko Unibertsitatea [Espagne] (UPV/EHU), Ikerbasque - Basque Foundation for Science, Université de Montréal (UdeM), Ruhr-Universität Bochum [Bochum], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Queen's University [Belfast] (QUB), The institute of cancer research [London], Université Laval [Québec] (ULaval), univOAK, Archive ouverte, German Research Foundation, and Collaborative Research Center 960 (Germany)
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RNA Polymerase I - genetics - metabolism ,Ecology ,Transcription Factors - metabolism ,Health, Toxicology and Mutagenesis ,Saccharomyces cerevisiae ,DNA ,Plant Science ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,SDG 3 - Good Health and Well-being ,RNA Polymerase I ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Saccharomyces cerevisiae - metabolism ,RNA Precursors ,Animals ,Humans ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,570 Biowissenschaften, Biologie ,ddc:570 ,Transcription Factors - Abstract
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble., This work was in part supported by the Emmy-Noether Programm (DFG grant no. EN 1204/1-1 to C Engel) of the German Research Council and Collaborative Research Center 960 (TP-A8 to C Engel).
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- 2022
89. R1 retrotransposons in the nucleolar organizers of Drosophila melanogaster are transcribed by RNA polymerase I upon heat shock.
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Raje, Himanshu S., Lieux, Molly E., and DiMario, Patrick J.
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RIBOSOMAL RNA , *RETROTRANSPOSONS , *DROSOPHILA melanogaster genetics , *RNA polymerase I , *PHOSPHOPROTEINS - Abstract
The ribosomal RNA genes (rDNA) of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200-300 tandem repeat rDNA units that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNA genes have R1 and/or R2 retrotransposons inserted at specific sites within their 28S regions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2 expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28S gene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 RNA junction and the expression of R1 elements within nucleolar rDNA rather than R1 elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1 elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5ʹ end of R1 where we find a consensus “pause button”, and that heat shock releases Pol I for read-through transcription farther into R1. [ABSTRACT FROM AUTHOR]
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- 2018
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90. Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos.
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Morovic, Martin, Østrup, Olga, Strejcek, Frantisek, Benc, Michal, Murin, Matej, Jedlickova, Katarina, Bartkova, Alexandra, Lucas-Hahn, Andrea, Pendovski, Lazo, and Laurincik, Jozef
- Abstract
Summary: The present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription. [ABSTRACT FROM AUTHOR]
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- 2018
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91. Copper-CX-5461: A novel liposomal formulation for a small molecule rRNA synthesis inhibitor.
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Leung, Ada W.y., Anantha, Malathi, Dragowska, Wieslawa H., Wehbe, Mohamed, and Bally, Marcel B.
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LIPOSOMES , *RIBOSOMAL RNA , *RNA polymerase I , *BRCA genes , *HEMATOLOGIC malignancies - Abstract
Abstract CX-5461 is currently in Phase I/II clinical trials for advanced hematologic malignancies and triple negative or BRCA-deficient breast cancer. The compound is currently administered to patients intravenously (i.v.) at low pH (3.5) due to solubility challenges. Reliance of low pH to enhance solubility of CX-5461 can adversely impact pharmacokinetics, biodistribution and therapeutic potential. We have addressed this solubility issue through a formulation method that relies on the interactions between CX-5461 and copper. Copper binds CX-5461 through the nitrogens of the pyrazine ring. Here, we describe synthesizing this copper-complexed CX-5461 (Cu(CX-5461)) within liposomes. CX-5461 was added to copper-containing liposomes and incubated at 60 °C for 30 min. The pharmacokinetics of CX-5461 was assessed in mice following a single i.v. injection at 30 mg/kg. Efficacy studies were completed in multiple subcutaneous mouse xenografts as well as in a bone marrow engraftment model of acute myeloid leukemia (AML). The novel Cu(CX-5461) formulation was stable at pH 7.4 and exhibited increased plasma circulation longevity, increasing the total exposure to CX5461 by an order of magnitude. Cu(CX-5461) was more active than CX-5461 in AML models in vivo. In HCT116-B46 and Capan-1 solid tumour models that are BRCA-deficient, the Cu(CX-5461) formulation engendered activity that was comparable to that of the low pH CX-5461 formulation. We have generated the first Cu(CX-5461) formulation suitable for i.v. administration that is more efficacious than the existing low-pH formulation in pre-clinical models of AML. The Cu(CX-5461) formulation may serve as an alternative formulation for CX-5461 in BRCA-deficient cancers. Graphical abstract Unlabelled Image Highlights • The phase I/II targeted agent CX-5461 is poorly soluble at physiological pH. • Metal-binding property of CX-5461 can be exploited for formulation strategies. • The first Cu(CX-5461) nanoformulation extends CX-5461 circulation lifetime. • Cu(CX-5461) is equally or more efficacious than the low pH CX-5461 formulation. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
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92. Structural basis of RNA polymerase I stalling at UV light-induced DNA damage.
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Sanz-Murillo, Marta, Jun Xu, Belogurov, Georgiy A., Calvo, Olga, Gil-Carton, David, Moreno-Morcillo, María, Dong Wang, and Fernández-Tornero, Carlos
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DNA damage , *RNA polymerases , *ENZYMES , *DIMERS , *TRANSCRIPTION factors - Abstract
RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPDinduced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
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93. Breaking the mold: structures of the RNA polymerase I transcription complex reveal a new path for initiation.
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Jackobel, Ashleigh J., Han, Yan, He, Yuan, and Knutson, Bruce A.
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RNA polymerase I , *GENETIC transcription , *INITIATION factors (Biochemistry) , *PROMOTERS (Genetics) , *PROTEIN structure - Abstract
While structures of the RNA polymerase (Pol) II initiation complex have been resolved and extensively studied, the Pol I initiation complex remained elusive. Here, we review the recent structural analyses of the yeast Pol I transcription initiation complex that reveal several unique and unexpected Pol I-specific properties. [ABSTRACT FROM AUTHOR]
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- 2018
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94. RNA polymerase I activation and hibernation: unique mechanisms for unique genes.
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Fernández-Tornero, Carlos
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HIBERNATION , *RNA polymerase I , *GENETIC transcription , *RIBOSOMAL DNA , *DIMERIZATION - Abstract
In yeast, transcription of ribosomal DNA (rDNA) by RNA polymerase I (Pol I) is regulated by unique mechanisms acting at the level of the enzyme. Under stress situations such as starvation, Pol I hibernates through dimerization. When growth conditions are restored, dimer disassembly and Rrn3 binding drive enzyme activation and subsequent recruitment to rDNA. [ABSTRACT FROM AUTHOR]
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- 2018
- Full Text
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95. The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis.
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Sriskanthadevan-Pirahas, Shrivani, Lee, Joshua, and Grewal, Savraj S.
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EPIDERMAL growth factor , *RENIN-angiotensin system , *DROSOPHILA , *RNA synthesis , *CELL proliferation , *TRANSCRIPTION factors - Abstract
The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. Here we report on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. We show that Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. We also find that activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. We show that Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, we find that TIF-IA-mediated rRNA synthesis is required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila , and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway. [ABSTRACT FROM AUTHOR]
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- 2018
- Full Text
- View/download PDF
96. Loss of Proteostasis Is a Pathomechanism in Cockayne Syndrome.
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Alupei, Marius Costel, Maity, Pallab, Esser, Philipp Ralf, Krikki, Ioanna, Tuorto, Francesca, Parlato, Rosanna, Penzo, Marianna, Schelling, Adrian, Laugel, Vincent, Montanaro, Lorenzo, Scharffetter-Kochanek, Karin, and Iben, Sebastian
- Abstract
Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS. • Cockayne syndrome cells show reduced translation fidelity • Elevated oxidative stress and misfolded proteins activate the unfolded protein response • The unfolded protein response suppresses Pol I activity in CS cells • Chemical chaperones alleviate ER stress and restore Pol activity in CS cells Cockayne syndrome is a devastating childhood progeria. Here, Alupei et al. show that cells from CS patients have reduced translation accuracy and elevated ROS, leading to generation of unstable proteins and activation of ER stress. Reducing ER stress by chemical chaperones in these cells rescues RNA polymerase I activity and protein synthesis. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
97. Impact of RNA polymerase I inhibitor CX-5461 on viral kinase-dependent and -independent cytomegalovirus replication.
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Westdorp, Kristen N. and Terhune, Scott S.
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HUMAN cytomegalovirus diseases , *RNA polymerase I , *VIRAL replication , *IMMUNOCOMPROMISED patients , *RIBOSOMAL DNA , *DNA structure - Abstract
Human cytomegalovirus (HCMV) infections cause congenital birth defects and disease in immunosuppressed individuals. Antiviral compounds can control infection yet their use is restricted due to concerns of toxicity and the emergence of drug resistant strains. We have evaluated the impact of an RNA Polymerase I (Pol I) inhibitor, CX-5461 on HCMV replication. CX-5461 inhibits Pol I-mediated ribosomal DNA transcription by binding G-quadruplex DNA structures and also activates cellular stress response pathways. The addition of CX-5461 at both early and late stages of the HCMV infection inhibited viral DNA synthesis and virus production. Interestingly, adding CX-5461 after the onset of viral DNA synthesis resulted in a greater reduction compared to continuous treatment starting early during infection. We observed an accompanying increase in cyclin-dependent kinase inhibitor p21 in infected cells treated late but not early which likely explains the differences. Our previous studies demonstrated the importance of p21 in the antiviral activity of the HCMV kinase inhibitor, maribavir. Addition of CX-5461 increased the anti-HCMV activity of maribavir. Our data demonstrate that CX-5461 inhibits HCMV replication and synergizes with maribavir to disrupt infection. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
98. Dynamics of a geminivirus-encoded pre-coat protein and host RNA-dependent RNA polymerase 1 in regulating symptom recovery in tobacco.
- Author
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Basu, Saumik, Kushwaha, Nirbhay Kumar, Singh, Ashish Kumar, Sahu, Pranav Pankaj, Kumar, R Vinoth, and Chakraborty, Supriya
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- *
TOBACCO disease & pest resistance , *PLANT viruses , *COAT proteins (Viruses) , *RNA polymerase I , *RNA interference , *GENETIC overexpression - Abstract
RNA silencing is an integral part of the cellular defense mechanisms in plants that act against virus infection. However, the specific role of RNA silencing and the interplay between host and virus components during recovery from geminivirus infection remains unknown. Hence, in this study we aimed to examine the mechanism behind the host-specific recovery of Nicotiana tabacum infected with Tomato leaf curl Gujarat virus (ToLCGV). Unlike Tomato leaf curl New Delhi virus (ToLCNDV), ToLCGV infection resulted in symptom remission in N. tabacum, and we found that this was mainly due to cross-talk between the pre-coat protein (encoded by the AV2 ORF) of the virus and the host RNA-silencing component RNA-dependent RNA polymerase 1 (encoded by NtRDR1) of N. tabacum. Moreover, apart from the AV2 mutant, other mutants of ToLCNDV developed severe symptoms on a transgenic NtRDR1-overexpression line of N. benthamiana. In contrast, inoculation with ToLCGV resulted in symptom remission, which was due to enhanced methylation of the ToLCGV promoter. Our study reveals a novel 'arms race' in which the pre-coat protein of ToLCNDV selectively blocks the recovery process through inhibiting host-specific RDR1-mediated antiviral silencing in tobacco. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
99. Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly.
- Author
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Smith, Marissa L., Cui, Weidong, Jackobel, Ashleigh J., Walker-Kopp, Nancy, and Knutson, Bruce A.
- Subjects
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SACCHAROMYCES cerevisiae , *RNA polymerase I , *PROMOTERS (Genetics) , *HISTONES , *GENETIC transcription - Abstract
RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli -based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
100. c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.
- Author
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Musso, Loana, Mazzini, Stefania, Rossini, Anna, Castagnoli, Lorenzo, Scaglioni, Leonardo, Artali, Roberto, Di Nicola, Massimo, Zunino, Franco, and Dallavalle, Sabrina
- Subjects
- *
RNA polymerase I , *MYC oncogenes , *CARRIER proteins , *NUCLEAR magnetic resonance , *ANTINEOPLASTIC agents , *MESSENGER RNA - Abstract
Background Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. Methods The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. Results and conclusions We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. General significance Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
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