Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

AbstractThe adjustment of transcription and translation rates to variable needs is of utmost importance for the fitness and survival of living cells. We have previously shown that the global transcription rate for RNA polymerase II is regulated differently in cells presenting symmetrical or asymmetrical cell division. The budding yeast Saccharomyces cerevisiae adopts a particular strategy to avoid that the smaller daughter cells increase their total mRNA concentration with every generation. The global mRNA synthesis rate lowers with a growing cell volume, but global mRNA stability increases. In this paper, we address what the solution is to the same theoretical problem for the RNA polymerase I synthesis rate. We find that the RNA polymerase I synthesis rate strictly depends on the copy number of its 35S rRNA gene. For cells with larger cell sizes, such as a mutant cln3 strain, the rDNA repeat copy number is increased by a mechanism based on a feed-back mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of the rRNA genes at the rDNA locus in a volume-dependent manner.

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Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

PLoS Genetics ◽

10.1371/journal.pgen.1009520 ◽

2021 ◽

Vol 17 (4) ◽

pp. e1009520

Author(s):

José E. Pérez-Ortín ◽

Adriana Mena ◽

Marina Barba-Aliaga ◽

Abhyudai Singh ◽

Sebastián Chávez ◽

...

Keyword(s):

Rna Polymerase ◽

Cell Volume ◽

Copy Number ◽

Rna Polymerase I ◽

Synthesis Rate ◽

Transcription Rate ◽

Rdna Repeat ◽

Repeat Copy Number ◽

Polymerase I ◽

Repeat Copy

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

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The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.9178-9188.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 9178-9188 ◽

Cited By ~ 104

Author(s):

Takehiko Kobayashi

Keyword(s):

Chromatin Immunoprecipitation ◽

Copy Number ◽

Replication Fork ◽

Rrna Genes ◽

Zinc Finger Motif ◽

Rdna Repeat ◽

Force Microscopy ◽

Repeat Copy Number ◽

Atomic Force ◽

Repeat Copy

ABSTRACT The replication fork barrier site (RFB) is an ∼100-bp DNA sequence located near the 3′ end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.

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Faculty Opinions recommendation of RNA polymerase I activators count and adjust ribosomal RNA gene copy number.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734776122.793556076 ◽

2019 ◽

Author(s):

Angela Taddei

Keyword(s):

Rna Polymerase ◽

Ribosomal Rna ◽

Copy Number ◽

Gene Copy Number ◽

Rna Polymerase I ◽

Gene Copy ◽

Ribosomal Rna Gene ◽

Polymerase I

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I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5809 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5809-5817 ◽

Cited By ~ 16

Author(s):

Jue Lin ◽

Volker M. Vogt

Keyword(s):

Rna Polymerase ◽

Homing Endonuclease ◽

Group I Intron ◽

Subcellular Fractionation ◽

Rna Polymerase I ◽

Rrna Genes ◽

Temperature Sensitive ◽

Group I ◽

Polymerase I ◽

Mutant Forms

ABSTRACT PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023–1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as theTetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.

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Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.114 ◽

1993 ◽

Vol 13 (1) ◽

pp. 114-122 ◽

Cited By ~ 59

Author(s):

Y Nogi ◽

R Yano ◽

J Dodd ◽

C Carles ◽

M Nomura

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Sequence ◽

Rna Polymerase ◽

Rna Polymerase I ◽

Rrna Genes ◽

Temperature Sensitive ◽

Polymerase I ◽

Temperature Sensitive Phenotype ◽

Null Mutants

We have previously isolated mutants of Saccharomyces cerevisiae that are primarily defective in transcription of 35S rRNA genes by RNA polymerase I and have identified genes (RRN1 to RRN9) involved in this process. We have now cloned the RRN4 gene by complementation of the temperature-sensitive phenotype of the rrn4-1 mutant and have determined its complete nucleotide sequence. The following results demonstrate that the RRN4 gene encodes the A12.2 subunit of RNA polymerase I. First, RRN4 protein expressed in Escherichia coli reacted with a specific antiserum against A12.2. Second, amino acid sequences of three tryptic peptides obtained from A12.2 were determined, and these sequences are found in the deduced amino acid sequence of the RRN4 protein. The amino acid sequence of the RRN4 protein (A12.2) is similar to that of the RPB9 (B12.6) subunit of yeast RNA polymerase II; the similarity includes the presence of two putative zinc-binding domains. Thus, A12.2 is a homolog of B12.6. We propose to rename the RRN4 gene RPA12. Deletion of RPA12 produces cells that are heat but not cold sensitive for growth. We have found that in such null mutants growing at permissive temperatures, the cellular concentration of A190, the largest subunit of RNA polymerase I, is lower than in the wild type. In addition, the temperature-sensitive phenotype of the rpa12 null mutants can be partially suppressed by RPA190 (the gene for A190) on multicopy plasmids. These results suggest that A12.2 plays a role in the assembly of A190 into a stable polymerase I structure.

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Nucleolin Is Required for RNA Polymerase I Transcription In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01584-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 937-948 ◽

Cited By ~ 76

Author(s):

Brenden Rickards ◽

S. J. Flint ◽

Michael D. Cole ◽

Gary LeRoy

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase I ◽

Rrna Genes ◽

Accessory Proteins ◽

Naked Dna ◽

Nucleolar Chromatin ◽

Polymerase I

ABSTRACT Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.

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Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole.

The Journal of Cell Biology ◽

10.1083/jcb.99.2.672 ◽

1984 ◽

Vol 99 (2) ◽

pp. 672-679 ◽

Cited By ~ 57

Author(s):

U Scheer ◽

B Hügle ◽

R Hazan ◽

K M Rose

Keyword(s):

Rna Polymerase ◽

Organizer Region ◽

Nucleolar Organizer Region ◽

Ribosomal Subunit ◽

Rna Polymerase I ◽

Rrna Genes ◽

Rrna Gene ◽

Linear Distribution ◽

Nucleolar Material ◽

Polymerase I

Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.

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In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.225 ◽

1996 ◽

Vol 133 (2) ◽

pp. 225-234 ◽

Cited By ~ 97

Author(s):

P Jordan ◽

M Mannervik ◽

L Tora ◽

M Carmo-Fonseca

Keyword(s):

Rna Polymerase ◽

Actinomycin D ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Rrna Genes ◽

Rrna Synthesis ◽

Rrna Transcription ◽

Polymerase I

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

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Tor Pathway Regulates Rrn3p-dependent Recruitment of Yeast RNA Polymerase I to the Promoter but Does Not Participate in Alteration of the Number of Active Genes

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0594 ◽

2004 ◽

Vol 15 (2) ◽

pp. 946-956 ◽

Cited By ~ 117

Author(s):

Jonathan A. Claypool ◽

Sarah L. French ◽

Katsuki Johzuka ◽

Kristilyn Eliason ◽

Loan Vu ◽

...

Keyword(s):

Rna Polymerase ◽

Stationary Phase ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

Synthesis Rate ◽

Transcription Rate ◽

Signaling System ◽

Tor Signaling ◽

Polymerase I ◽

Active Genes

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

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