Chihiro Tsutsumi

Selective elimination of messenger RNA prevents an incidence of untimely meiosis.

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.

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint

Abstract. The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Meiosis-specific noncoding RNA mediates robust pairing of homologous chromosomes in meiosis.

Find Your Partner Gametes generally have haploid genomes, so that when they fuse during fertilization, they reconstitute a diploid organism. Meiosis is required to make haploid gametes, which involves a reduction division where two homologous chromosomes first pair and are then segregated away from each other. Working in the fission yeast Schizosaccharomyces pombe, Ding et al. (p. 732; see the Perspective by Dernburg) explored how homologous chromosomes recognized each other and found that a non-coding RNA locus, sme2, helps to drive the pairing of homologous chromosomes. An RNA transcript helps to bring together homologous chromosomes during cell division. 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.

Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation

A screen identifies two more bouquet proteins required for meiotic telomere clustering: Bqt4 anchors the telomeres, whereas Bqt3 protects Bqt4 from degradation.

Two-step, extensive alterations in the transcriptome from G0 arrest to cell division in Schizosaccharomyces pombe.

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 ~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 ~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.

Localization of gene products using a chromosomally tagged GFP‐fusion library in the fission yeast Schizosaccharomyces pombe

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.

A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex.

Mutations in the human Tsc1 and Tsc2 genes predispose to tuberous sclerosis complex (TSC), a disorder characterized by the wide spread of benign tumors. Tsc1 and Tsc2 proteins form a complex and serve as a GTPase-activating protein (GAP) for Rheb, a GTPase regulating a downstream kinase, mTOR. The genome of Schizosaccharomyces pombe contains tsc1+ and tsc2+, homologs of human Tsc1 and Tsc2, respectively. In this study we analyzed the gene expression profile on a genomewide scale and found that deletion of either tsc1+ or tsc2+ affects gene induction upon nitrogen starvation. Three hours after nitrogen depletion genes encoding permeases and genes required for meiosis are less induced. Under the same condition, retrotransposons, G1-cyclin (pas1+), and inv1+ are more induced. We also demonstrate that a mutation (cpp1-1) in a gene encoding a β-subunit of a farnesyltransferase can suppress most of the phenotypes associated with deletion of tsc1+ or tsc2+. When a mutant of rhb1+ (homolog of human Rheb), which bypasses the requirement of protein farnesylation, was expressed, the cpp1-1 mutation could no longer suppress, indicating that deficient farnesylation of Rhb1 contributes to the suppression. On the basis of these results, we discuss TSC pathology and possible improvement in chemotherapy for TSC.

Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement

Abstract.We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement. Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase α. We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division. Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1. Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division. We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion.

Mediator Directs Co-transcriptional Heterochromatin Assembly by RNA Interference-Dependent and -Independent Pathways

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.

Distinctive Responses to Nitrogen Starvation in the Dominant Active Mutants of the Fission Yeast Rheb GTPase

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.