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.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals RNA Polymerase I-Promoted HIS4Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (2) ◽  
pp. 665-675 ◽  
Author(s):  
Hsiu-Jung Lo ◽  
Han-Kuei Huang ◽  
Thomas F. Donahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Mutant Form ◽  
Wild Type ◽  
Pol I ◽  
Chromosome Iii ◽  
Polymerase I

ABSTRACT The HIS4 gene in Saccharomyces cerevisiaewas put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. Therhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-typeHIS4 mRNA. Its 3′ end maps to the HIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l, is bicistronic. It encodes the HIS4coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3′ end of rhis4l maps to the predicted 3′ end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5′ end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5′-3′ exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-typeHIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1Δ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5′ cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

2012 ◽  
Vol 302 (10) ◽  
pp. C1523-C1530 ◽  
Author(s):  
Ferdinand von Walden ◽  
Vandre Casagrande ◽  
Ann-Kristin Östlund Farrants ◽  
Gustavo A. Nader
Keyword(s):  
Skeletal Muscle ◽  
Mechanical Loading ◽  
Muscle Hypertrophy ◽  
Rrna Synthesis ◽  
Rdna Transcription ◽  
Hypertrophic Response ◽  
Skeletal Muscle Hypertrophy ◽  
Pol I ◽  
Upstream Binding Factor ◽  
Polymerase I

The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

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.

scholarly journals The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eva Torreira ◽  
Jaime Alegrio Louro ◽  
Irene Pazos ◽  
Noelia González-Polo ◽  
David Gil-Carton ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Mutational Analysis ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rdna Transcription ◽  
Pol I ◽  
Polymerase I

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

scholarly journals Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin

2004 ◽  
Vol 24 (4) ◽  
pp. 1791-1798 ◽  
Author(s):  
Ralf Strohner ◽  
Attila Németh ◽  
Karl P. Nightingale ◽  
Ingrid Grummt ◽  
Peter B. Becker ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Active Role ◽  
Rna Polymerase I ◽  
In Vivo Studies ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription Terminator ◽  
Polymerase I

ABSTRACT The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.

scholarly journals Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

2006 ◽  
Vol 26 (16) ◽  
pp. 5957-5968 ◽  
Author(s):  
Tatiana B. Panova ◽  
Kostya I. Panov ◽  
Jackie Russell ◽  
Joost C. B. M. Zomerdijk
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Rna Polymerase I ◽  
Positive Effects ◽  
Pol I ◽  
Polymerase I

ABSTRACT Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Iβ isoform and not with Pol Iα. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Iβ-associated CK2 can phosphorylate topoisomerase IIα in Pol Iβ, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

scholarly journals Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene.

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325 ◽  
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals Association of Yeast RNA Polymerase I with a Nucleolar Substructure Active in Rrna Synthesis and Processing

2000 ◽  
Vol 149 (3) ◽  
pp. 575-590 ◽  
Author(s):  
Stephan Fath ◽  
Philipp Milkereit ◽  
Alexandre V. Podtelejnikov ◽  
Nicolas Bischler ◽  
Patrick Schultz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rrna Synthesis ◽  
Small Nucleolar Rnas ◽  
Rdna Transcription ◽  
Nucleolar Proteins ◽  
Pol I ◽  
Synthesis And Processing ◽  
Polymerase I ◽  
Nucleolar Rnas ◽  
Processing Factors

A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry. When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p. The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA. Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units. Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I. Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing.

scholarly journals Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

2018 ◽  
Author(s):  
Tommy Darrière ◽  
Michael Pilsl ◽  
Marie-Kerguelen Sarthou ◽  
Adrien Chauvier ◽  
Titouan Genty ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Pol I ◽  
Polymerase I ◽  
Extragenic Suppressors

AbstractMost 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 wild-type 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.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

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