scholarly journals In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.

1993 ◽  
Vol 13 (2) ◽  
pp. 928-933 ◽  
Author(s):  
S M Vallett ◽  
M Brudnak ◽  
M Pellegrini ◽  
H W Weber
Keyword(s):  
Phorbol Ester ◽  
Growth Regulation ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription System ◽  
Pol I ◽  
Drosophila Cells

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum

1993 ◽  
Vol 13 (2) ◽  
pp. 928-933
Author(s):  
S M Vallett ◽  
M Brudnak ◽  
M Pellegrini ◽  
H W Weber
Keyword(s):  
Phorbol Ester ◽  
Growth Regulation ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription System ◽  
Pol I ◽  
Drosophila Cells

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

scholarly journals Dichotomous Impact of Myc on rRNA Gene Activation and Silencing in B Cell Lymphomagenesis

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3009
Author(s):  
Gaurav Joshi ◽  
Alexander Otto Eberhardt ◽  
Lisa Lange ◽  
René Winkler ◽  
Steve Hoffmann ◽  
...  
Keyword(s):  
B Cell ◽  
Gene Activation ◽  
Cpg Methylation ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Highly Active ◽  
Pol I ◽  
Polymerase I ◽  
Transcriptional Output

A major transcriptional output of cells is ribosomal RNA (rRNA), synthesized by RNA polymerase I (Pol I) from multicopy rRNA genes (rDNA). Constitutive silencing of an rDNA fraction by promoter CpG methylation contributes to the stabilization of these otherwise highly active loci. In cancers driven by the oncoprotein Myc, excessive Myc directly stimulates rDNA transcription. However, it is not clear when during carcinogenesis this mechanism emerges, and how Myc-driven rDNA activation affects epigenetic silencing. Here, we have used the Eµ-Myc mouse model to investigate rDNA transcription and epigenetic regulation in Myc-driven B cell lymphomagenesis. We have developed a refined cytometric strategy to isolate B cells from the tumor initiation, promotion, and progression phases, and found a substantial increase of both Myc and rRNA gene expression only in established lymphoma. Surprisingly, promoter CpG methylation and the machinery for rDNA silencing were also strongly up-regulated in the tumor progression state. The data indicate a dichotomous role of oncogenic Myc in rDNA regulation, boosting transcription as well as reinforcing repression of silent repeats, which may provide a novel angle on perturbing Myc function in cancer cells.

scholarly journals In Exponentially Growing Saccharomyces cerevisiae Cells, rRNA Synthesis Is Determined by the Summed RNA Polymerase I Loading Rate Rather than by the Number of Active Genes

2003 ◽  
Vol 23 (5) ◽  
pp. 1558-1568 ◽  
Author(s):  
Sarah L. French ◽  
Yvonne N. Osheim ◽  
Francesco Cioci ◽  
Masayasu Nomura ◽  
Ann L. Beyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Exponential Growth ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Transcription Rate ◽  
Initiation Rate ◽  
Pol I ◽  
Genes Encoding ◽  
Active Genes

ABSTRACT Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes (∼143 in this case) and a strain in which the rRNA gene number was reduced to ∼42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was ∼50 ± 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 ± 29 polymerases/gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of ∼60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

scholarly journals Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

2008 ◽  
Vol 28 (21) ◽  
pp. 6709-6719 ◽  
Author(s):  
Robert D. Hontz ◽  
Sarah L. French ◽  
Melanie L. Oakes ◽  
Prasad Tongaonkar ◽  
Masayasu Nomura ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Rrna Synthesis ◽  
Synthesis Rate ◽  
Wild Type ◽  
Rdna Transcription ◽  
Rdna Genes ◽  
Pol I ◽  
Polymerase I ◽  
I Cells

ABSTRACT Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ∼70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

scholarly journals Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Benjamin Albert ◽  
Jorge Perez-Fernandez ◽  
Isabelle Léger-Silvestre ◽  
Olivier Gadal
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Chromatin Structure ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Elongation Factors ◽  
Pol I ◽  
Polymerase I

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

scholarly journals Stop-and-Go: Dynamics of Nucleolar Transcription During the Cell Cycle

2019 ◽  
Vol 12 ◽  
pp. 251686571984909 ◽  
Author(s):  
Aishwarya Iyer-Bierhoff ◽  
Ingrid Grummt
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Histone H2a ◽  
Rdna Transcription ◽  
Chromatin Dynamics ◽  
Chromatin Compaction ◽  
Pol I

Entry into mitosis correlates with nucleolar disassembly and shutdown of ribosomal RNA (rRNA) gene (rDNA) transcription. In telophase, nucleoli reform and transcription is reactivated. The molecular mechanisms underlying the dynamics of nucleolar transcription during the cell cycle are manifold. Although mitotic inactivation of the RNA polymerase I (Pol I) transcription machinery by posttranslational modifications has been extensively studied, little is known about the structure of rDNA chromatin during progression through mitosis. Methylation of histone H2A at glutamine 104 (H2AQ104me), a dedicated nucleolar histone modification, is lost in prometaphase, leading to chromatin compaction, which enforces mitotic repression of rRNA genes. At telophase, restoration of H2AQ104me is required for the activation of transcription. H2AQ104 methylation and chromatin dynamics are regulated by fibrillarin (FBL) and the NAD+-dependent nucleolar deacetylase sirtuin 7 (SIRT7). Deacetylation of FBL is required for the methylation of H2AQ104 and high levels of rDNA transcription during interphase. At the entry into mitosis, nucleoli disassemble and FBL is hyperacetylated, leading to loss of H2AQ104me, chromatin compaction, and shutdown of Pol I transcription. These results reveal that reversible acetylation of FBL regulates methylation of nucleolar H2AQ104, thereby reinforcing oscillation of Pol I transcription during the cell cycle.

scholarly journals Heat shock selectively inhibits ribosomal RNA gene transcription and down-regulates E1BF/Ku in mouse lymphosarcoma cells

1996 ◽  
Vol 317 (3) ◽  
pp. 689-695 ◽  
Author(s):  
Kalpana GHOSHAL ◽  
Samson T. JACOB
Keyword(s):  
Heat Shock ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Binding Activity ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Pol I ◽  
Mouse Lymphosarcoma

The effect of heat shock on RNA polymerase I (pol I)-directed transcription of the rRNA gene was studied in S-100 extract derived from mouse lymphosarcoma cells, and by in vivo labelling of rRNA. Exposure of cells to 42 °C for 2 h resulted in complete inhibition of rRNA synthesis in vivo. Pol I transcription was inhibited by 50% within 2 h of heat shock and was abolished after 3 h exposure at 42 °C. Under this condition, the core-promoter-binding activity of the factor (CPBF) that modulates pol I transcription was unaffected. In contrast, the promoter-binding activity of enhancer-1-binding factor, a protein related to the Ku autoantigen, which is involved in pol I transcription initiation, was reduced by 50 and 90% after 2 and 3 h of heat shock respectively. Western-blot analysis with antibodies specific for the two subunits of Ku protein showed the absence of p72 subunit after 3 h of heat shock. Under this condition, pol II transcription from the adenovirus major late promoter and pol III transcription of 5 S RNA gene remained unaffected. Mixing experiments ruled out the possibility that the inhibition of transcription was due to activation of nucleases or other inhibitors. This is the first report to show selective down-regulation of pol I transcription in vitro by heat shock and of the potential involvement of a pol I transcription factor in this process.

scholarly journals Yeast RNA Polymerase I Enhancer Is Dispensable for Transcription of the Chromosomal rRNA Gene and Cell Growth, and Its Apparent Transcription Enhancement from Ectopic Promoters Requires Fob1 Protein

2001 ◽  
Vol 21 (16) ◽  
pp. 5541-5553 ◽  
Author(s):  
Hobert Wai ◽  
Katsuki Johzuka ◽  
Loan Vu ◽  
Kristilyn Eliason ◽  
Takehiko Kobayashi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase ◽  
Hot Spot ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Enhancer Region ◽  
Pol I ◽  
Polymerase I

ABSTRACT At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.

scholarly journals Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1156
Author(s):  
Jiachen Xuan ◽  
Kezia Gitareja ◽  
Natalie Brajanovski ◽  
Elaine Sanij
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Therapy ◽  
Dna Damage Response ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Pol I ◽  
Clinical Investigations ◽  
Damage Response ◽  
Synthesis And Processing

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli

1991 ◽  
Vol 99 (4) ◽  
pp. 759-767
Author(s):  
M. Thiry ◽  
G. Goessens
Keyword(s):  
Tumor Cell ◽  
Condensed Chromatin ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Dense Fibrillar Component ◽  
Ehrlich Tumor ◽  
Granular Component ◽  
Fibrillar Component

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.

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