[Show abstract][Hide abstract]ABSTRACT: In ribosome biogenesis, a large fraction of ribosomes is used for producing ribosomal proteins themselves. Here, we applied simulation and experimentation to determine what fraction of ribosomes should be allocated for the synthesis of ribosomal proteins to optimize cellular economy for growth. We define the 'r-fraction' as the fraction of mRNA of the ribosomal protein genes out of the total mRNA, and we simulated the effect of the r-fraction on the number of ribosomes. We then empirically measured the amount of protein and RNA in fission yeast cells cultured with high and low nitrogen sources. In the cells cultured with a low nitrogen source, the r-fraction decreased from 0.46 to 0.42 with a 40% reduction of rRNA, but the reduction of the total protein was smaller at 30%. These results indicate that the r-fraction is internally controlled to optimize the efficiency of protein synthesis at a limited cellular cost.
Full-text · Article · Oct 2015 · Scientific Reports
[Show abstract][Hide abstract]ABSTRACT: Mediator is required for heterochromatin silencing at the pericentromere. (A) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto non-selective media (N/S), medium without uracil (-Ura), medium with 5-fluoroorotic acid (5-FOA), and medium without adenine (-Ade) to assay ura4+ and ade6+expression. Note that PMGS (EMMS-NH4Cl (nitrogen), +L-glutamic acid, monosodium, as nitrogen) plates were used as N/S plates. (B) Schematic of Med8-K9 and Med31-H1 proteins. med8-K9 contains a point mutation (C540T) causing C-terminal truncation of the Med8 protein (Q135*) (top). The C-terminal residues 176–200 of Med8 (Med8C) are the predicted interface for interaction with Med18 , . med31-H1 contains a point mutation (T230A) causing C-terminal truncation of the Med31 protein (L77*) (bottom). Residues 22–77 of Med31 are the predicted interface for interaction with Med7, a core Mediator subunit belonged to the middle domain. (C) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto non-selective media (N/S), medium with 5-FOA, and medium with a limited amount of adenine (Low Ade) to assay for the presence of imr1L::ura4+ and otr1R::ade6+.
[Show abstract][Hide abstract]ABSTRACT: Mediator mutations cause accumulation of centromeric RNA. (A) Schematic of fission yeast centromere 1. Locations of the ura4+ and ade6+ reporters inserted within the pericentromeric region are shown (imr1L::ura4+ and otr1R::ade6+). Black bars indicate the location of probes used for northern analysis. (B) Northern analysis of pericentromeric transcripts in wild-type and mutant cells. Analysis was performed using oligonucleotide probes against dh and dg forward (Fw) and reverse (Rv) strands, imr1L::ura4+ and otr1R::ade6+ forward strand transcripts. rRNA and tRNA were used as loading controls.
[Show abstract][Hide abstract]ABSTRACT: Effect of Mediator disruption on euchromatic genes. Venn diagram showing the number of transcripts whose expression levels were increased (up) or decreased (down) >1.5-fold in mutants compared to the wild type. The p-value was calculated using Fisher's exact test. (A) Transcripts of med18Δ-w (left circles) vs. med20Δ-w (right circles) mutants. (B) Transcripts of med18Δ-p (left circles) vs. med20Δ-p (right circles) mutants.
[Show abstract][Hide abstract]ABSTRACT: Conversion rates of white/pink epiclones. Stability assay of variegation phenotypes. Conversion rates (percentage of cells that convert the epigenetic state per generation) were measured using indicated strains. w indicates the white epiclones and p indicates the pink epiclones. Cells were grown for several generations. The rates were measured as described in supplemental experimental procedures.
[Show abstract][Hide abstract]ABSTRACT: Northern analysis of siRNA in the Mediator mutants med8-K9 and med31-H1. Analyses were performed with RNA isolated from the indicated strains (panel A: single deletion mutants, panel B: single and double deletion mutants of dcr1 and med18) using oligonucleotide probes specific for dg/dh centromeric repeats. tRNA was used as a loading control.
[Show abstract][Hide abstract]ABSTRACT: Med20 localizes the pericentromeric heterochromatin with RNAPII in a cell cycle-dependent manner. (A, B) ChIP-qPCR analyses for RNAPII and Med20-5FLAG were performed every 30 minutes after release from G2/M block by cdc25-22 mutation (see Supplemental Experimental Procedures). Enrichment at dh repeats relative to the adh1 promoter region (adh1 pro) is shown. Error bars show the standard error of the mean (n = 3). (C) Septation index (percentage of cells with division septum) was measured to monitor cell cycle progression after release. In a block and release experiment, the peak of septation (90 to 120 min) indicates S phase. (D) RT-PCR of dh transcripts was performed as described in A and B. Transcripts derived from constitutively expressed act1 served as controls. “No RT” indicates that no reverse transcriptase was added in the reaction. Numbers under the panels of dh and act1 indicate the increase in transcript relative to the values at 0 min (dh) and 60 min (act1), respectively.
[Show abstract][Hide abstract]ABSTRACT: Mediator is required for transcriptional activation in heterochromatin. ChIP analysis of RNAPII at cenH dh repeats or kint2::ura4+ relative to input WCE in the indicated strains. Anti-RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation was used. Error bars represent the standard error of the mean (n = 3). P values were determined using a two-sided Student's t-test.
[Show abstract][Hide abstract]ABSTRACT: Mediator negatively regulates RNAPII in the compromised heterochromatin. ChIP analysis of RNAPII at dh repeats relative to the gene free region in the indicated strains. Error bars show the standard error of the mean (n = 3).
[Show abstract][Hide abstract]ABSTRACT: Heterochromatin at the pericentromeric repeats in fission yeast is assembled and spread by an RNAi-dependent mechanism, which is coupled with the transcription of non-coding RNA from the repeats by RNA polymerase II. In addition, Rrp6, a component of the nuclear exosome, also contributes to heterochromatin assembly and is coupled with non-coding RNA transcription. The multi-subunit complex Mediator, which directs initiation of RNA polymerase II-dependent transcription, has recently been suggested to function after initiation in processes such as elongation of transcription and splicing. However, the role of Mediator in the regulation of chromatin structure is not well understood. We investigated the role of Mediator in pericentromeric heterochromatin formation and found that deletion of specific subunits of the head domain of Mediator compromised heterochromatin structure. The Mediator head domain was required for Rrp6-dependent heterochromatin nucleation at the pericentromere and for RNAi-dependent spreading of heterochromatin into the neighboring region. In the latter process, Mediator appeared to contribute to efficient processing of siRNA from transcribed non-coding RNA, which was required for efficient spreading of heterochromatin. Furthermore, the head domain directed efficient transcription in heterochromatin. These results reveal a pivotal role for Mediator in multiple steps of transcription-coupled formation of pericentromeric heterochromatin. This observation further extends the role of Mediator to co-transcriptional chromatin regulation.
[Show abstract][Hide abstract]ABSTRACT: Pairing and recombination of homologous chromosomes are essential for ensuring reductional segregation in meiosis. However,
the mechanisms by which chromosomes recognize their homologous partners are poorly understood. Here, we report that the sme2 gene encodes a meiosis-specific noncoding RNA that mediates homologous recognition in the fission yeast Schizosaccharomyces pombe. The sme2 locus shows robust pairing from early in meiotic prophase. The sme2 RNA transcripts accumulate at their respective gene loci and greatly enhance pairing of homologous loci: Deletion of the
sme2 sequence eliminates this robust pairing, whereas transposition to other chromosomal sites confers robust pairing at those
ectopic sites. Thus, we propose that RNA transcripts retained on the chromosome play an active role in recognition of homologous
chromosomes for pairing.
[Show abstract][Hide abstract]ABSTRACT: In many organisms, telomeres cluster to form a bouquet arrangement of chromosomes during meiotic prophase. Previously, we reported that two meiotic proteins, Bqt1 and -2, are required for tethering telomeres to the spindle pole body (SPB) during meiotic prophase in fission yeast. This study has further identified two novel, ubiquitously expressed inner nuclear membrane (INM) proteins, Bqt3 and -4, which are required for bouquet formation. We found that in the absence of Bqt4, telomeres failed to associate with the nuclear membranes in vegetative cells and consequently failed to cluster to the SPB in meiotic prophase. In the absence of Bqt3, Bqt4 protein was degraded during meiosis, leading to a phenotype similar to that of the bqt4-null mutant. Collectively, these results show that Bqt4 anchors telomeres to the INM and that Bqt3 protects Bqt4 from protein degradation. Interestingly, the functional integrity of telomeres is maintained even when they are separated from the nuclear envelope in vegetative cells.
Full-text · Article · Nov 2009 · The Journal of Cell Biology
[Show abstract][Hide abstract]ABSTRACT: Rheb, a Ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in the regulation of cell growth depending on extracellular conditions. Rhb1 (a fission yeast homolog of Rheb) regulates amino acid uptake as well as response to nitrogen starvation. In this study, we generated two mutants, rhb1-DA4 and rhb1-DA8, and characterized them genetically. The V17A mutation within the G1 box defined for the Ras-like GTPases was responsible for rhb1-DA4 and Q52R I76F within the switch II domain for rhb1-DA8. In fission yeast, two events--the induction of the meiosis-initiating gene mei2+ and cell division without cell growth--are a typical response to nitrogen starvation. Under nitrogen-rich conditions, Rheb stimulates Tor kinase, which, in turn, suppresses the response to nitrogen starvation. While amino acid uptake was prevented by both rhb1-DA4 and rhb1-DA8 in a dominant fashion, the response to nitrogen starvation was prevented only by rhb1-DA4. rhb1-DA8 thereby allowed genetic dissection of the Rheb-dependent signaling cascade. We postulate that the signaling cascade may branch below Rhb1 or Tor2 and regulate the amino acid uptake and response to nitrogen starvation independently.
[Show abstract][Hide abstract]ABSTRACT: We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3'-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP-fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm.
[Show abstract][Hide abstract]ABSTRACT: Body cells in multicellular organisms are in the G0 state, in which cells are arrested and terminally differentiated. To understand how the G0 state is maintained, the genes that are specifically expressed or repressed in G0 must be identified, as they control G0. In the fission yeast Schizosaccharomyces pombe, haploid cells are completely arrested under nitrogen source starvation with high viability. We examined the global transcriptome of G0 cells and cells on the course to resume vegetative growth. Approximately 20% of the transcripts of approximately 5000 genes increased or decreased more than fourfold in the two-step transitions that occur prior to replication. Of the top 30 abundant transcripts in G0, 23 were replaced by ribosome- and translation-related transcripts in the dividing vegetative state. Eight identified clusters with distinct alteration patterns of approximately 2700 transcripts were annotated by Gene Ontology. Disruption of 53 genes indicated that nine of them were necessary to support the proper G0 state. These nine genes included two C2H2 zinc finger transcription factors, a cyclin-like protein implicated in phosphorylation of RNA polymerase II, two putative autophagy regulators, a G-protein activating factor, and two CBS domain proteins, possibly involved in AMP-activated kinase.
[Show abstract][Hide abstract]ABSTRACT: Imbalances of gene expression in aneuploids, which contain an abnormal number of chromosomes, cause a variety of growth and developmental defects. Aneuploid cells of the fission yeast Schizosaccharomyces pombe are inviable, or very unstable, during mitotic growth. However, S. pombe haploid cells bearing minichromosomes derived from the chromosome 3 can grow stably as a partial aneuploid. To address biological consequences of aneuploidy, we examined the gene expression profiles of partial aneuploid strains using DNA microarray analysis. The expression of genes in disomic or trisomic cells was found to increase approximately in proportion to their copy number. We also found that some genes in the monosomic regions of partial aneuploid strains increased their expression level despite there being no change in copy number. This change in gene expression can be attributed to increased expression of the genes in the disomic or trisomic regions. However, even in an aneuploid strain that bears a minichromosome containing no protein coding genes, genes located within about 50 kb of the telomere showed similar increases in expression, indicating that these changes are not a secondary effect of the increased gene dosage. Examining the distribution of the heterochromoatin protein Swi6 using DNA microarray analysis, we found that binding of Swi6 within ~50 kb from the telomere occurred less in partial aneuploid strains compared to euploid strains. These results suggest that additional chromosomes in aneuploids could lead to imbalances in gene expression through changes in distribution of heterochromatin as well as in gene dosage.
Full-text · Article · Feb 2007 · Cell Structure and Function
[Show abstract][Hide abstract]ABSTRACT: Much remains unknown about the molecular regulation of meiosis. Here we show that meiosis-specific transcripts are selectively removed if expressed during vegetative growth in fission yeast. These messenger RNAs contain a cis-acting region--which we call the DSR--that confers this removal via binding to a YTH-family protein Mmi1. Loss of Mmi1 function severely impairs cell growth owing to the untimely expression of meiotic transcripts. Microarray analysis reveals that at least a dozen such meiosis-specific transcripts are eliminated by the DSR-Mmi1 system. Mmi1 remains in the form of multiple nuclear foci during vegetative growth. At meiotic prophase these foci precipitate to a single focus, which coincides with the dot formed by the master meiosis-regulator Mei2. A meiotic arrest due to the loss of the Mei2 dot is released by a reduction in Mmi1 activity. We propose that Mei2 turns off the DSR-Mmi1 system by sequestering Mmi1 to the dot and thereby secures stable expression of meiosis-specific transcripts.