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

PLoS Genetics ◽  
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.

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

2019 ◽  
Author(s):  
José E. Pérez-Ortín ◽  
Adriana Mena ◽  
Marina Barba-Aliaga ◽  
Rebeca Alonso-Monge ◽  
Abhyudai Singh ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cell Volume ◽  
Copy Number ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Synthesis Rate ◽  
Rdna Repeat ◽  
Repeat Copy Number ◽  
Polymerase I ◽  
Repeat Copy

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.

scholarly journals 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

2004 ◽  
Vol 15 (2) ◽  
pp. 946-956 ◽  
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.

scholarly journals Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

1990 ◽  
Vol 10 (5) ◽  
pp. 2049-2059 ◽  
Author(s):  
M Wittekind ◽  
J M Kolb ◽  
J Dodd ◽  
M Yamagishi ◽  
S Mémet ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Polymerase I ◽  
Northern Analysis ◽  
Rrna Synthesis ◽  
Synthesis Rate ◽  
Conditional Expression ◽  
Regulation Model ◽  
Polymerase I ◽  
Precursor Mrna

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).

scholarly journals A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

2014 ◽  
Vol 35 (3) ◽  
pp. 544-554 ◽  
Author(s):  
Inswasti Cahyani ◽  
Andrew G. Cridge ◽  
David R. Engelke ◽  
Austen R. D. Ganley ◽  
Justin M. O'Sullivan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Intergenic Region ◽  
Copy Number ◽  
Rna Polymerase I ◽  
Intergenic Sequence ◽  
Polymerase I ◽  
Rdna Copy ◽  
Rdna Copy Number

The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within theSaccharomyces cerevisiaegenome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e.,RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within theRPA135-tK(CUU)Pintergenic region that is involved in the interaction. We demonstrate that theRPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not governRPA135transcription. Instead, replacement of a 605-bp region within theRPA135-tK(CUU)Pintergenic region results in a reduction in theRPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between theRPA135-tK(CUU)Pand rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome.

scholarly journals Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

2018 ◽  
Author(s):  
Sam Cutler ◽  
Laura J Lee ◽  
Toshio Tsukiyama
Keyword(s):  
Chromatin Remodeling ◽  
Ribosomal Rna ◽  
Ribosomal Dna ◽  
Chromatin Structure ◽  
Copy Number ◽  
Rdna Repeat ◽  
Repeat Copy Number ◽  
Dependent Processes ◽  
Rna Genes ◽  
Repeat Copy

AbstractIn the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.Author SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the “ribosomal DNA locus”, a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.

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

2003 ◽  
Vol 23 (24) ◽  
pp. 9178-9188 ◽  
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.

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis

1990 ◽  
Vol 10 (5) ◽  
pp. 2049-2059
Author(s):  
M Wittekind ◽  
J M Kolb ◽  
J Dodd ◽  
M Yamagishi ◽  
S Mémet ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Polymerase I ◽  
Northern Analysis ◽  
Rrna Synthesis ◽  
Synthesis Rate ◽  
Conditional Expression ◽  
Regulation Model ◽  
Polymerase I ◽  
Precursor Mrna

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).

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