The RNA binding protein Csx1 promotes sexual differentiation in schizosaccharomyces pombe

Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source. One of the key regulatory proteins in fission yeast sexual differentiation is the transcription factor Ste11. Ste11 regulates the transcription of many genes required for the initial steps of conjugation and meiosis, and its deficiency leads to sterility. Ste11 activity is mainly regulated at two levels: phosphorylation and abundance of its mRNA. Csx1 is an RNA binding protein that we have previously described to bind and regulate the turnover rate of the mRNA encoding the transcription factor Atf1 in the presence of oxidative stress. We have observed that Csx1-deficient cells have defects in sexual differentiation and are partially sterile. We investigated how Csx1 is regulating this process in S. pombe. Csx1 associates with ste11 + mRNA and cells lacking Csx1 are sterile with a reduced amount of ste11 + mRNA. Overexpression of ste11 + mRNA completely rescues the mating deficiencies of csx1Δ cells. Here, we present a novel mechanism of ste11 + mRNA positive regulation through the activity of Csx1, an RNA binding protein that also have key functions in the response to oxidative stress in fission yeast. This finding opens interesting question about the possible coordination of sexual differentiation and oxidative stress response in eukaryotes and the role of RNA binding proteins in the adaptation to environmental signals.Spanish Ministry of Science and Innovation (BFU2006-01767, BFU2009-09116); Madrid GovernmentPeer Reviewe

The RNA Binding Protein Csx1 Promotes Sexual Differentiation in Schizosaccharomyces pombe

Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source

Csx1 binds <i>ste11⁺</i> mRNA and regulates its abundance.

<p>Quantitative real-time PCR analysis of <i>ste11⁺</i> mRNA (A) and <i>atf1⁺</i> mRNA (B) in homothallic wild type and <i>csx1</i>Δ strains in minimal media (EMM) and in the absence of nitrogen source (EMM-N) for 6 hours. Bars indicate standard error. C. Binding of Csx1 to <i>ste11⁺</i> mRNA. Cell extract was obtained (IN) and TAP immunoprecipitation (IP) was performed with homothallic wild type and <i>csx1:TAP</i> strains in minimal media (EMM) and in the absence of ammonium source (EMM-N) for 1 hour and 3 hours. RNA was isolated and cDNA generated by reverse transcription. <i>ste11⁺</i> mRNA was amplified by PCR and monitored by agarose electrophoresis (455 bps). D. Strains and treatment conditions were identical to those described in C. Binding of Csx1:TAP to <i>ste11⁺</i> mRNA was measured by reverse transcription followed by quantitative PCR. Actin mRNA was used as control. The graph represents <i>ste11⁺</i> mRNA relative levels in input samples and after Csx1:TAP purification (IP).</p

Response to Arsenate treatment in Schizosaccharomyces pombe and the role of its Arsenate reductase activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.Spanish Science and Innovation Ministry (CTQ2008-01031/BQU, BFU2006/01767, BFU2009/09116)Peer Reviewe

Csx1 is required for sexual differentiation in fission yeast.

<p>Morphology of homothallic wild type (A) and <i>csx1</i>Δ (B) strains after 48 hours in ME at 24°C. Pictures were taken using Nomarski filter. C. Mating percentage (conjugation/meiosis) reached by homothallic wild type and <i>csx1</i>Δ strains after 48 hours in ME at 24°C. Bars indicate standard error. D. Meiotic percentage reached by diploid h⁻/h⁺<i>csx1⁺</i>/<i>csx1⁺</i> and h⁻/h⁺<i>csx1</i>Δ/<i>csx1</i>Δ strains after 48 hours in ME at 24°C. Bars indicate standard error.</p

G1 arrest upon nitrogen starvation in several strains.

<p>Homothallic wild type, <i>spc1</i>Δ, <i>ste11</i>Δ, <i>csx1</i>Δ strains were incubated in EMM-N for up to 24 hours and samples were taken at the indicated times. DNA relative amount was estimated by the fluorescent signal amount emitted by propidium iodide and the flow citometry histograms represented.</p

Overproduction of s<i>te11⁺</i> mRNA rescues <i>csx1</i>Δ sterility.

<p>A. Homothallic wild type and <i>csx1</i>Δ strains transformed with pREP1 and pREP1-Ste11 plasmids were plated in minimal media without nitrogen or thiamine and incubated for 48 hours at 24°C. Afterwards mating percentage was calculated. Bars indicate standard error. B. Homothallic <i>csx1</i>Δ strain was transformed with pREP1:Csx1 plasmid and mating percentage measured in minimal media with/without nitrogen source or promoter repressing thiamine (B1). In C and D, homothallic <i>csx1</i>Δ strains overexpressing Ste11 or Csx1, were grown in EMM-N media for 6 hours with or without thiamine (+B1/−B1) and <i>ste11⁺</i> mRNA (C) or of <i>atf1⁺</i> mRNA (D) measured by RT-qPCR.</p

Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast

Spc1 MAPK pathway and the response to arsenate.

<p>A. Serial dilutions of wild type, <i>wis1</i>Δ, <i>mcs4</i>Δ, <i>wis4</i>Δ, <i>win1-1</i> and <i>wis4</i>Δ <i>win1-1</i> strains were plated in rich media (YES) or rich media containing 50 µM sodium arsenate. Pictures were taken after incubation at 30°C for 48 hours. B. Western blotting of purified Spc1 extracts from wild type, <i>wis1</i>Δ, <i>wis1-AA</i>, <i>win1-1, wis4</i>Δ, and <i>win1-1 wis4</i>Δ treated with 100 µM sodium arsenate for 0 to 30 minutes. Antibodies against phosphorylated p38 were used. As a control, antibodies against HA epitope were used. C. Western blotting of purified Spc1 extracts from wild type, <i>wis1</i>Δ, <i>win1-1 wis4</i>Δ, <i>win1-1 wis4</i>Δ <i>pyp1</i>Δ and <i>win1-1 wis4</i>Δ <i>pyp2</i>Δ treated with 100 µM sodium arsenate for 0 to 30 minutes. Antibodies against phosphorylated p38 were used. As a control, antibodies against HA epitope were used.</p