A central role for TOR signalling in a yeast model for juvenile CLN3 disease

Yeasts provide an excellent genetically tractable eukaryotic system for investigating the function of genes in their biological context, and are especially relevant for those conserved genes that cause disease. We study the role of btn1, the orthologue of a human gene that underlies an early onset neurodegenerative disease (juvenile CLN3 disease, neuronal ceroid lipofuscinosis (NCLs) or Batten disease) in the fission yeast Schizosaccharomyces pombe. A global screen for genetic interactions with btn1 highlighted a conserved key signalling hub in which multiple components functionally relate to this conserved disease gene. This signalling hub includes two major mitogen-activated protein kinase (MAPK) cascades, and centers on the Tor kinase complexes TORC1 and TORC2. We confirmed that yeast cells modelling CLN3 disease exhibit features consistent with dysfunction in the TORC pathways, and showed that modulating TORC function leads to a comprehensive rescue of defects in this yeast disease model. The same pathways may be novel targets in the development of therapies for the NCLs and related diseases

A central role for TOR signalling in a yeast model for juvenile CLN3 disease

Yeasts provide an excellent genetically tractable eukaryotic system for investigating the function of genes in their biological context, and are especially relevant for those conserved genes that cause disease. We study the role of btn1, the orthologue of a human gene that underlies an early onset neurodegenerative disease (juvenile CLN3 disease, neuronal ceroid lipofuscinosis (NCLs) or Batten disease) in the fission yeast Schizosaccharomyces pombe. A global screen for genetic interactions with btn1 highlighted a conserved key signalling hub in which multiple components functionally relate to this conserved disease gene. This signalling hub includes two major mitogen-activated protein kinase (MAPK) cascades, and centers on the Tor kinase complexes TORC1 and TORC2. We confirmed that yeast cells modelling CLN3 disease exhibit features consistent with dysfunction in the TORC pathways, and showed that modulating TORC function leads to a comprehensive rescue of defects in this yeast disease model. The same pathways may be novel targets in the development of therapies for the NCLs and related diseases

The copper transport-associated protein Ctr4 can form prion-like epigenetic determinants in Schizosaccharomyces pombe

Prions are protein-based infectious entities associated with fatal brain diseases in animals, but also modify a range of host-cell phenotypes in the budding yeast, Saccharomyces cerevisiae. Many questions remain about the evolution and biology of prions. Although several functionally distinct prion-forming proteins exist in S. cerevisiae, [HET-s] of Podospora anserina is the only other known fungal prion. Here we investigated prion-like, protein-based epigenetic transmission in the fission yeast Schizosaccharomyces pombe. We show that S. pombe cells can support the formation and maintenance of the prion form of the S. cerevisiae Sup35 translation factor [PSI+], and that the formation and propagation of these Sup35 aggregates is inhibited by guanidine hydrochloride, indicating commonalities in prion propagation machineries in these evolutionary diverged yeasts. A proteome-wide screen identified the Ctr4 copper transporter subunit as a putative prion with a predicted prion-like domain. Overexpression of the ctr4 gene resulted in large Ctr4 protein aggregates that were both detergent and proteinase-K resistant. Cells carrying such [CTR+] aggregates showed increased sensitivity to oxidative stress, and this phenotype could be transmitted to aggregate-free [ctr–] cells by transformation with [CTR+] cell extracts. Moreover, this [CTR+] phenotype was inherited in a non-Mendelian manner following mating with naïve [ctr–] cells, but intriguingly the [CTR+] phenotype was not eliminated by guanidine-hydrochloride treatment. Thus, Ctr4 exhibits multiple features diagnostic of other fungal prions and is the first example of a prion in fission yeast. These findings suggest that transmissible protein-based determinants of traits may be more widespread among fungi

Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast.

Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

Background: The genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays.|Results: We designed PCR primers to amplify specific probes (180-500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (similar to13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts.|Conclusions: This paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Cuf2 Is a Novel Meiosis-Specific Regulatory Factor of Meiosis Maturation

Meiosis is the specialized form of the cell cycle by which diploid cells produce the haploid gametes required for sexual reproduction. Initiation and progression through meiosis requires that the expression of the meiotic genes is precisely controlled so as to provide the correct gene products at the correct times. During meiosis, four temporal gene clusters are either induced or repressed by a cascade of transcription factors

The <i>cuf2</i>Δ/<i>cuf2</i>Δ mutant is defective in FSM formation.

<p><i>A</i>, Both wild-type diploid (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells expressing GFP-Psy1 were synchronously induced to undergo azygotic meiosis. At the 9 h time point, FSM formation was monitored by detecting the GFP-Psy1 signal by fluorescence microscopy. When GFP-Psy1 localized to 4 circular FSM structures, these FSMs were classified as normal (i). When GFP-Psy1 localized to either lesser or greater than 4 circular FSMs or shmoo-like structures, FSMs were classified as abnormal (ii, iii and iv). <i>B</i>, Histograms showing the percentages of each normal (i) and abnormal (ii, iii, iv) FSM structure in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells, as well as in a diploid <i>cuf2Δ/cuf2Δ</i> disruption strain in which wild-type copies of the <i>cuf2⁺-GFP</i> fusion gene were reintegrated. <i>C</i>, Typical images of FSM structures 9 h after meiotic induction in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells (top panels). Each strain had previously been transformed with pJK210GFP-Psy1, which encodes GFP-Psy1 that is used as an FSM-resident marker. Hoechst 33342 staining was used to visualize the chromosomal DNA (middle panels). The merged images of the GFP-Psy1 and the Hoechst 33342 dye are shown in the bottom panels. Anucleated FSM structures, or unpackaged nuclei, are indicated by the white arrows.</p

Analysis of Cuf2-GFP localization during both meiosis and sporulation.

<p>The Cuf2-GFP fluorescence signal (center left) was observed at different stages of meiosis following the azygotic meiotic induction of a <i>h⁺/h⁻ cuf2Δ/cuf2Δ cuf2⁺-GFP/cuf2⁺-GFP</i> strain. Once induced, azygotic meiotic cells were differentiated under basal conditions. Cuf2-GFP was observed at each extremity of the cell at the end of the first meiotic division (MI) that is to say at anaphase I. Cuf2-GFP followed the segregation of chromatids until late in anaphase II when the second meiotic division (MII) occurred in early anaphase II. The Cuf2-GFP fluorescence signal was detected during the FSM formation (i.e. at the end of meiosis), but disappeared during spore formation. Cells at different stages of meiosis were stained using Hoechst 33342 to visualize the DNA (centre right). The merged images are shown in the far right panels. Nomarski optics (far left) were used to monitor cell morphology.</p