scholarly journals 5-Fluorouracil Enhances Exosome-Dependent Accumulation of Polyadenylated rRNAs

2004 ◽  
Vol 24 (24) ◽  
pp. 10766-10776 ◽  
Author(s):  
Feng Fang ◽  
Jason Hoskins ◽  
J. Scott Butler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
General Result ◽  
Rna Processing ◽  
Ribosome Biogenesis ◽  
Dna Microarrays ◽  
Rrna Processing ◽  
Genome Wide ◽  
Genes Encoding ◽  
Nuclear Exosome

ABSTRACT The antimetabolite 5-fluorouracil (5FU) is a widely used chemotherapeutic for the treatment of solid tumors. Although 5FU slows DNA synthesis by inhibiting the ability of thymidylate synthetase to produce dTMP, the drug also has significant effects on RNA metabolism. Recent genome-wide assays for 5FU-induced haploinsufficiency in Saccharomyces cerevisiae identified genes encoding components of the RNA processing exosome as potential targets of the drug. In this report, we used DNA microarrays to analyze the effect of 5FU on the yeast transcriptome and found that the drug causes the accumulation of polyadenylated fragments of the 27S rRNA precursor and that defects in the nuclear exoribonuclease Rrp6p enhance this effect. The size distribution of these RNAs and their sensitivity to Rrp6p suggest that they are normally degraded by the nuclear exosome and a 5′-3′ exoribonuclease. Consistent with this hypothesis, 5FU inhibits the growth of RRP6 mutants with defects in the degradation function of the enzyme and it interferes with the degradation of an rRNA precursor. The detection of poly(A)+ pre-RNAs in strains defective in various steps in ribosome biogenesis suggests that the production of poly(A)+ pre-rRNAs may be a general result of defects in rRNA processing. These findings suggest that 5FU inhibits an exosome-dependent surveillance pathway that degrades polyadenylated precursor rRNAs.

scholarly journals Intricate regulation of ribosome biogenesis genes in response to mTORC1 signaling

2021 ◽  
Author(s):  
Sanjay Kumar ◽  
Muneera Mashkoor ◽  
Priya Balamurugan ◽  
Anne Grove
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Target Genes ◽  
Common Ancestor ◽  
Gene Promoters ◽  
Rrna Processing ◽  
Processing Factor ◽  
Mtorc1 Signaling ◽  
Genes Encoding

SummaryGenes encoding ribosomal proteins are repressed in response to inhibition of mTORC1. In Saccharomyces cerevisiae, this involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which can mediate mTORC1 inhibition by acting as a repressor. Ifh1p and Crf1p derive from a common ancestor, which may have acted as both an activator and a repressor. We report here that UTP22 and RRP7, which encodes another pre-rRNA processing factor, are controlled by mTORC1; both gene promoters are bound by Ifh1p, which dissociates on mTORC1 inhibition. Notably, Crf1p acts as an activator as evidenced by reduced expression in a crf1Δ strain. By contrast, Crf1p is required to repress expression of HMO1, which encodes a cofactor involved in communicating mTORC1 activity to target genes. Our data therefore indicate that Crf1p exhibits the dual repressor/activator functions of the Ifh1p-Crf1p ancestor.

scholarly journals Genome-Wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Posttranscriptional Control of Ribosome Biogenesis Factors

2004 ◽  
Vol 24 (12) ◽  
pp. 5534-5547 ◽  
Author(s):  
Jörg Grigull ◽  
Sanie Mnaimneh ◽  
Jeffrey Pootoolal ◽  
Mark D. Robinson ◽  
Timothy R. Hughes
Keyword(s):  
Heat Stress ◽  
Microarray Data ◽  
Mrna Stability ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Transcriptional Response ◽  
Rrna Synthesis ◽  
Ribosome Assembly ◽  
Genome Wide ◽  
Genes Encoding

ABSTRACT Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

scholarly journals Identification of Genes That Function in the Biogenesis and Localization of Small Nucleolar RNAs in Saccharomyces cerevisiae

2008 ◽  
Vol 28 (11) ◽  
pp. 3686-3699 ◽  
Author(s):  
Hui Qiu ◽  
Julia Eifert ◽  
Ludivine Wacheul ◽  
Marc Thiry ◽  
Adam C. Berger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Large Scale ◽  
Cajal Body ◽  
Temperature Sensitive ◽  
Small Nucleolar Rnas ◽  
Animal Cells ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Nucleolar Rnas

ABSTRACT Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

scholarly journals Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1758-1771 ◽  
Author(s):  
A. de Morgan ◽  
L. Brodsky ◽  
Y. Ronin ◽  
E. Nevo ◽  
A. Korol ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Exponential Phase ◽  
Yeast Cells ◽  
Genome Wide ◽  
Dna Turnover ◽  
Genomic Scale ◽  
Dna Maintenance

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2′-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

Comparison of the yeast and human nuclear exosome complexes

2012 ◽  
Vol 40 (4) ◽  
pp. 850-855 ◽  
Author(s):  
Katherine E. Sloan ◽  
Claudia Schneider ◽  
Nicholas J. Watkins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Processing ◽  
Eukaryotic Cells ◽  
Multiprotein Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Surveillance ◽  
Mature Transcript ◽  
Nuclear Exosome ◽  
And Function ◽  
Rna Maturation

Most RNAs in eukaryotic cells are produced as precursors that undergo processing at the 3′ and/or 5′ end to generate the mature transcript. In addition, many transcripts are degraded not only as part of normal recycling, but also when recognized as aberrant by the RNA surveillance machinery. The exosome, a conserved multiprotein complex containing two nucleases, is involved in both the 3′ processing and the turnover of many RNAs in the cell. A series of factors, including the TRAMP (Trf4–Air2–Mtr4 polyadenylation) complex, Mpp6 and Rrp47, help to define the targets to be processed and/or degraded and assist in exosome function. The majority of the data on the exosome and RNA maturation/decay have been derived from work performed in the yeast Saccharomyces cerevisiae. In the present paper, we provide an overview of the exosome and its role in RNA processing/degradation and discuss important new insights into exosome composition and function in human cells.

scholarly journals Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (4) ◽  
pp. 1496-1509 ◽  
Author(s):  
Amine Nourani ◽  
Francois Robert ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Transcription Elongation ◽  
Coding Region ◽  
Elongation Factors ◽  
Chromatin Dynamics ◽  
Coding Regions ◽  
Genome Wide ◽  
Genes Encoding ◽  
Approaches To Study

ABSTRACT Spt2/Sin1 is a DNA binding protein with HMG-like domains that has been suggested to play a role in chromatin-mediated transcription in Saccharomyces cerevisiae. Previous studies have suggested models in which Spt2 plays an inhibitory role in the initiation of transcription of certain genes. In this work, we have taken several approaches to study Spt2 in greater detail. Our results have identified previously unknown genetic interactions between spt2Δ and mutations in genes encoding transcription elongation factors, including members of the PAF and HIR/HPC complexes. In addition, genome-wide and gene-specific chromatin immunoprecipitation analyses suggest that Spt2 is primarily associated with coding regions in a transcription-dependent fashion. Furthermore, our results show that Spt2, like other elongation factors, is required for the repression of transcription from a cryptic promoter within a coding region and that Spt2 is also required for repression of recombination within transcribed regions. Finally, we provide evidence that Spt2 plays a role in regulating the levels of histone H3 over transcribed regions. Taken together, our results suggest a direct link for Spt2 with transcription elongation, chromatin dynamics, and genome stability.

scholarly journals Dynamic Distribution of SeqA Protein across the Chromosome of Escherichia coli K-12

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
María Antonia Sánchez-Romero ◽  
Stephen J. W. Busby ◽  
Nigel P. Dyer ◽  
Sascha Ott ◽  
Andrew D. Millard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Microarrays ◽  
Nucleotide Metabolism ◽  
Entire Genome ◽  
Content Type ◽  
Genome Wide ◽  
Genes Encoding ◽  
K 12

ABSTRACTThe bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. InEscherichia coliK-12, the single replication originoriCis a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequestersoriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entireEscherichia coliK-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in thetermacrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.IMPORTANCEDNA replication in bacteria is a highly regulated process. In many bacteria, a protein called SeqA plays a key role by binding to newly replicated DNA. Thus, at the origin of DNA replication, SeqA binding blocks premature reinitiation of replication rounds. Although most investigators have focused on the role of SeqA at replication origins, it has long been suspected that SeqA has a more pervasive role. In this study, we describe how we have been able to identify scores of targets, across the entireEscherichia colichromosome, to which SeqA binds. Using synchronously growing cells, we show that the distribution of SeqA between these targets alters as replication of the chromosome progresses. This suggests that sequential changes in SeqA distribution orchestrate a program of gene expression that ensures coordinated DNA replication and cell division.

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