H2O2 stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4.

In fission yeast, the stress-activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H(2)O(2) but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H(2)O(2), resulting in the full-length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4.

Loss of regulators of vacuolar ATPase function and ceramide synthesis results in multidrug sensitivity in Schizosaccharomyces pombe.

We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H(+))-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.

Distinct regulatory proteins control the graded transcriptional response to increasing H(2)O(2) levels in fission yeast Schizosaccharomyces pombe.

The signaling pathways that sense adverse stimuli and communicate with the nucleus to initiate appropriate changes in gene expression are central to the cellular stress response. Herein, we have characterized the role of the Sty1 (Spc1) stress-activated mitogen-activated protein kinase pathway, and the Pap1 and Atf1 transcription factors, in regulating the response to H(2)O(2) in the fission yeast Schizosaccharomyces pombe. We find that H(2)O(2) activates the Sty1 pathway in a dose-dependent manner via at least two sensing mechanisms. At relatively low levels of H(2)O(2), a two component-signaling pathway, which feeds into either of the two stress-activated mitogen-activated protein kinase kinase kinases Wak1 or Win1, regulates Sty1 phosphorylation. In contrast, at high levels of H(2)O(2), Sty1 activation is controlled predominantly by a two-component independent mechanism and requires the function of both Wak1 and Win1. Individual transcription factors were also found to function within a limited range of H(2)O(2) concentrations. Pap1 activates target genes primarily in response to low levels of H(2)O(2), whereas Atf1 primarily controls the transcriptional response to high concentrations of H(2)O(2). Our results demonstrate that S. pombe uses a combination of stress-responsive regulatory proteins to gauge and effect the appropriate transcriptional response to increasing concentrations of H(2)O(2).

Hypoxia-driven splicing into noncoding isoforms regulates the DNA damage response.

Tumour hypoxia is associated with poor patient outcome and resistance to therapy. It is accompanied by widespread changes in gene expression mediated largely through the transcription factors HIF1/2/3α. Hypoxia impacts on multiple pathways throughout the cell and has widespread effects on phenotype. Here we use sample-specific annotation approaches to determine the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. Using in vivo data generated from a time course in reduced oxygenation we identified genome-wide switching between coding and noncoding isoforms, including a significant number of components of the DNA damage response pathway. Notably, HDAC6, a master regulator of the cytotoxic response, and TP53BP1, which sits at the nexus of the double-strand break repair pathway, both underwent a marked transition towards an intron-retention pattern with a concomitant decline in protein levels. These transitions from coding to noncoding isoforms were recapitulated in a large and independent cohort of 499 colorectal samples taken from The Cancer Genome Atlas (TCGA). The set of altered genes was enriched for multiple components of the Fanconi Anaemia, nucleotide excision and double-strand break repair pathways, and together correlating with tumour status at last contact. Altogether, these data demonstrate a new role for hypoxia-driven alternative splicing in regulating DNA damage response, and highlight the importance of considering alternative splicing as a critical factor in our understanding of human disease.

A global non-coding RNA system modulates fission yeast protein levels in response to stress.

Non-coding RNAs (ncRNAs) are frequent and prevalent across the taxa. Although individual non-coding loci have been assigned a function, most are uncharacterized. Their global biological significance is unproven and remains controversial. Here we investigate the role played by ncRNAs in the stress response of Schizosaccharomyces pombe. We integrate global proteomics and RNA sequencing data to identify a systematic programme in which elevated antisense RNA arising both from ncRNAs and from 3'-overlapping convergent gene pairs is directly associated with substantial reductions in protein levels throughout the genome. We describe an extensive array of ncRNAs with trans associations that have the potential to influence multiple pathways. Deletion of one such locus reduces levels of atf1, a transcription factor downstream of the stress-activated mitogen-activated protein kinase (MAPK) pathway, and alters sensitivity to oxidative stress. These non-coding transcripts therefore regulate specific stress responses, adding unanticipated information-processing capacity to the MAPK signalling system.

Fission yeast MAP kinase Sty1 is recruited to stress-induced genes.

The stress-induced expression of many fission yeast genes is dependent upon the Sty1 mitogen-activated protein kinase (MAPK) and Atf1 transcription factor. Atf1 is phosphorylated by Sty1 yet this phosphorylation is not required for stress-induced gene expression, suggesting another mechanism exists whereby Sty1 activates transcription. Here we show that Sty1 associates with Atf1-dependent genes and is recruited to both their promoters and coding regions. This occurs in response to various stress conditions coincident with the kinetics of the activation of Sty1. Association with promoters is not a consequence of increased nuclear accumulation of Sty1 nor does it require the phosphorylation of Atf1. However, recruitment is completely abolished in a mutant lacking Sty1 kinase activity. Both Atf1 and its binding partner Pcr1 are required for association of Sty1 with Atf1-dependent promoters, suggesting that this heterodimer must be intact for optimal recruitment of the MAPK. However, many Atf1-dependent genes are still expressed in a pcr1Delta mutant but with significantly delayed kinetics, thus providing an explanation for the relatively mild stress sensitivity displayed by pcr1Delta. Consistent with this delay, Sty1 and Atf1 cannot be detected at these promoters in this condition, suggesting that their association with chromatin is weak or transient in the absence of Pcr1.