Evolution of the early spliceosomal complex-from constitutive to regulated splicing

Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5' and 3' splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.This work and APC was funded by the Spanish Ministerio de Economia y Competitividad (MINECO), PLAN E and Feder and by Unidad de Excelencia María de Maeztu, grant numbers: BFU2018-PGC2018-097248-B-I00 and CEX2018-000792-M

Cells lacking pfh1, a fission yeast homolog of mammalian frataxin protein, display constitutive activation of the iron starvation response

Friedreich ataxia is a genetic disease caused by deficiencies in frataxin. This protein has homologs not only in higher eukaryotes but also in bacteria, fungi, and plants. The function of this protein is still controversial. We have identified a frataxin homolog in fission yeast, and we have analyzed whether its depletion leads to any of the phenotypes observed in other organisms. Cells deleted in pfh1 are sensitive to growth under aerobic conditions, display increased levels of total iron, hallmarks of oxidative stress such as protein carbonylation, decreased aconitase activity, and lower levels of oxygen consumption compared with wild-type cells. This mitochondrial protein seems to be important for iron and/or reactive oxygen species homeostasis. We have analyzed the proteome of cells devoid of Pfh1, and we determined that gene products up- and down-regulated upon iron depletion in wild-type cells are constitutively misregulated in this mutant. Because of the particular signaling pathway components governing the iron starvation response in fission yeast, our experiments suggest that cells lacking Pfh1 display a decrease of cytosolic available iron that triggers activation of Grx4, the common regulator of the iron starvation gene expression program. Our Schizosaccharomyces pombe Δpfh1 strain constitutes a new and useful model system to study Friedreich ataxia.This work was supported in part by Spanish Ministry of Science and Innovation Grants BFU2009-06933 and BFU2012-32045, PLAN E, FEDER, by Spanish Program Consolider-Ingenio 2010 Grant CSD 2007-0020, and by Generalitat de Catalunya (Spain) Grant SGR2009-196 (to E. H.)

Gcn5-mediated acetylation at MBF-regulated promoters induces the G1/S transcriptional wave

In fission yeast, MBF-dependent transcription is inactivated at the end of S phase through a negative feedback loop that involves the co-repressors, Yox1 and Nrm1. Although this repression system is well known, the molecular mechanisms involved in MBF activation remain largely unknown. Compacted chromatin constitutes a barrier to activators accessing promoters. Here, we show that chromatin regulation plays a key role in activating MBF-dependent transcription. Gcn5, a part of the SAGA complex, binds to MBF-regulated promoters through the MBF co-activator Rep2 in a cell cycle-dependent manner and in a reverse correlation to the binding of the MBF co-repressors, Nrm1 or Yox1. We propose that the co-repressors function as physical barriers to SAGA recruitment onto MBF promoters. We also show that Gcn5 acetylates specific lysine residues on histone H3 in a cell cycle-regulated manner. Furthermore, either in a gcn5 mutant or in a strain in which histone H3 is kept in an unacetylated form, MBF-dependent transcription is downregulated. In summary, Gcn5 is required for the full activation and correct timing of MBF-regulated gene transcription.Spanish Ministerio de Economia y Competitividad, PLAN E, and Feder [BFU2015-66347, PGC2018-097248-B-I00]; MEIONet [BFU2015-71786-REDT]; Unidad de Excelencia Maria de Maeztu [MDM-2014-0370]; ICREA Academia Award (Generalitat de Catalunya) (to E.H.). Funding for open access charge: Spanish Ministerio de Economia y Competitividad [BFU2015-66347, PGC2018-097248-B-I00]

Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity

BACKGROUND: Hydrogen peroxide (H2O2) is generated as a by-product of metabolic reactions during oxygen use by aerobic organisms, and can be toxic or participate in signaling processes. Cells, therefore, need to be able to sense and respond to H2O2 in an appropriate manner. This is often accomplished through thiol switches: Cysteine residues in proteins that can act as sensors, and which are both scarce and finely tuned. Bacteria and eukaryotes use different types of such sensors-either a one-component (OxyR) or two-component (Pap1-Tpx1) redox relay, respectively. However, the biological significance of these two different signaling modes is not fully understood, and the concentrations and peroxides driving those types of redox cascades have not been determined, nor the intracellular H2O2 levels linked to toxicity. Here we elucidate the characteristics, rates, and dynamic ranges of both systems. RESULTS: By comparing the activation of both systems in fission yeast, and applying mathematical equations to the experimental data, we estimate the toxic threshold of intracellular H2O2 able to halt aerobic growth, and the temporal gradients of extracellular to intracellular peroxides. By calculating both the oxidation rates of OxyR and Tpx1 by peroxides, and their reduction rates by the cellular redoxin systems, we propose that, while Tpx1 is a sensor and an efficient H2O2 scavenger because it displays fast oxidation and reduction rates, OxyR is strictly a H2O2 sensor, since its reduction kinetics are significantly slower than its oxidation by peroxides, and therefore, it remains oxidized long enough to execute its transcriptional role. We also show that these two paradigmatic H2O2-sensing models are biologically similar at pre-toxic peroxide levels, but display strikingly different activation behaviors at toxic doses. CONCLUSIONS: Both Tpx1 and OxyR contain thiol switches, with very high reactivity towards peroxides. Nevertheless, the fast reduction of Tpx1 defines it as a scavenger, and this efficient recycling dramatically changes the Tpx1-Pap1 response to H2O2 and connects H2O2 sensing to the redox state of the cell. In contrast, OxyR is a true H2O2 sensor but not a scavenger, being partially insulated from the cellular electron donor capacity.This work is supported by the Ministerio de Economía y Competitividad (Spain), PLAN E, and FEDER (BFU2015-68350-P to EH), by Fundação para a Ciência e a Tecnologia (Portugal;, project UID/MULTI/00612/2013 to FA), and by Generalitat de Catalunya (Spain; 2014-SGR-154 to EH and JA). AD is the recipient of a pre-doctoral fellowship from Generalitat de Catalunya (Spain). EH is the recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain)

Gcn5-mediated acetylation at MBF-regulated promoters induces the G1/S transcriptional wave

In fission yeast, MBF-dependent transcription is inactivated at the end of S phase through a negative feedback loop that involves the co-repressors, Yox1 and Nrm1. Although this repression system is well known, the molecular mechanisms involved in MBF activation remain largely unknown. Compacted chromatin constitutes a barrier to activators accessing promoters. Here, we show that chromatin regulation plays a key role in activating MBF-dependent transcription. Gcn5, a part of the SAGA complex, binds to MBF-regulated promoters through the MBF co-activator Rep2 in a cell cycle-dependent manner and in a reverse correlation to the binding of the MBF co-repressors, Nrm1 or Yox1. We propose that the co-repressors function as physical barriers to SAGA recruitment onto MBF promoters. We also show that Gcn5 acetylates specific lysine residues on histone H3 in a cell cycle-regulated manner. Furthermore, either in a gcn5 mutant or in a strain in which histone H3 is kept in an unacetylated form, MBF-dependent transcription is downregulated. In summary, Gcn5 is required for the full activation and correct timing of MBF-regulated gene transcription.Spanish Ministerio de Economia y Competitividad, PLAN E, and Feder [BFU2015-66347, PGC2018-097248-B-I00]; MEIONet [BFU2015-71786-REDT]; Unidad de Excelencia Maria de Maeztu [MDM-2014-0370]; ICREA Academia Award (Generalitat de Catalunya) (to E.H.). Funding for open access charge: Spanish Ministerio de Economia y Competitividad [BFU2015-66347, PGC2018-097248-B-I00]

Genome-wide screening of regulators of catalase expression: role of a transcription complex and histone and tRNA modification complexes on adaptation to stress

In response to environmental cues, the mitogen-activated protein kinase Sty1-driven signaling cascade activates hundreds of genes to induce a robust anti-stress cellular response in fission yeast. Thus, upon stress imposition Sty1 transiently accumulates in the nucleus where it up-regulates transcription through the Atf1 transcription factor. Several regulators of transcription and translation have been identified as important to mount an integral response to oxidative stress, such as the Spt-Ada-Gcn5-acetyl transferase or Elongator complexes, respectively. With the aim of identifying new regulators of this massive gene expression program, we have used a GFP-based protein reporter and screened a fission yeast deletion collection using flow cytometry. We find that the levels of catalase fused to GFP, both before and after a threat of peroxides, are altered in hundreds of strains lacking components of chromatin modifiers, transcription complexes, and modulators of translation. Thus, the transcription elongation complex Paf1, the histone methylase Set1-COMPASS, and the translation-related Trm112 dimers are all involved in full expression of Ctt1-GFP and in wild-type tolerance to peroxidesThis work was supported by the Spanish Ministry of Science and Innovation (BFU2012-32045 (to E.H.) and BFU2012-31939 (to J.A.), PLAN E and FEDER, and by 2014-SGR-154 from Generalitat de Catalunya (Spain) (to E.H. and J.A.)

Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity

BACKGROUND: Hydrogen peroxide (H2O2) is generated as a by-product of metabolic reactions during oxygen use by aerobic organisms, and can be toxic or participate in signaling processes. Cells, therefore, need to be able to sense and respond to H2O2 in an appropriate manner. This is often accomplished through thiol switches: Cysteine residues in proteins that can act as sensors, and which are both scarce and finely tuned. Bacteria and eukaryotes use different types of such sensors-either a one-component (OxyR) or two-component (Pap1-Tpx1) redox relay, respectively. However, the biological significance of these two different signaling modes is not fully understood, and the concentrations and peroxides driving those types of redox cascades have not been determined, nor the intracellular H2O2 levels linked to toxicity. Here we elucidate the characteristics, rates, and dynamic ranges of both systems. RESULTS: By comparing the activation of both systems in fission yeast, and applying mathematical equations to the experimental data, we estimate the toxic threshold of intracellular H2O2 able to halt aerobic growth, and the temporal gradients of extracellular to intracellular peroxides. By calculating both the oxidation rates of OxyR and Tpx1 by peroxides, and their reduction rates by the cellular redoxin systems, we propose that, while Tpx1 is a sensor and an efficient H2O2 scavenger because it displays fast oxidation and reduction rates, OxyR is strictly a H2O2 sensor, since its reduction kinetics are significantly slower than its oxidation by peroxides, and therefore, it remains oxidized long enough to execute its transcriptional role. We also show that these two paradigmatic H2O2-sensing models are biologically similar at pre-toxic peroxide levels, but display strikingly different activation behaviors at toxic doses. CONCLUSIONS: Both Tpx1 and OxyR contain thiol switches, with very high reactivity towards peroxides. Nevertheless, the fast reduction of Tpx1 defines it as a scavenger, and this efficient recycling dramatically changes the Tpx1-Pap1 response to H2O2 and connects H2O2 sensing to the redox state of the cell. In contrast, OxyR is a true H2O2 sensor but not a scavenger, being partially insulated from the cellular electron donor capacity.This work is supported by the Ministerio de Economía y Competitividad (Spain), PLAN E, and FEDER (BFU2015-68350-P to EH), by Fundação para a Ciência e a Tecnologia (Portugal;, project UID/MULTI/00612/2013 to FA), and by Generalitat de Catalunya (Spain; 2014-SGR-154 to EH and JA). AD is the recipient of a pre-doctoral fellowship from Generalitat de Catalunya (Spain). EH is the recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain)

TOR and MAP kinase pathways synergistically regulate autophagy in response to nutrient depletion in fission yeast

Data de publicació electrònica: 23-06-2021General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired.Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.We thank Kaoru Takegawa for kindly providing plasmid pGFP-Atg8 in pREP41 and strain SK1, and Akio Nakashima and Fuyuhiko Tamanoi for providing AN0175, AN0179 and JUP1350 strains. We thank Li-Lin Du for helpful discussions. This work was supported by the Ministerio de Ciencia, Innovación y Universidades, PLAN E and FEDER (Spain) (PGC2018-093920-B-I00 to E.H.). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Unidad de Excelencia María de Maeztu, funded by the AEI (Spain) (CEX2018-000792-M). R.B. is recipient of a FPI contract from the Ministerio de Ciencia, Innovación y Universidades (Spain). E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain)

Stress-induced cell depolarization through the MAP kinase-Cdc42 axis

General stress responses, which sense environmental or endogenous signals, aim at promoting cell survival and fitness during adverse conditions. In eukaryotes, mitogen-activated protein (MAP) kinase-driven cascades trigger a shift in the cell's gene expression program as a cellular adaptation to stress. Here, we review another aspect of activated MAP kinase cascades reported in fission yeast: the transient inhibition of cell polarity in response to oxidative stress. The phosphorylation by a stress-activated MAP kinase of regulators of the GTPase cell division cycle 42 (Cdc42) causes a transient inhibition of polarized cell growth. The formation of growth sites depends on limiting and essential polarity components. We summarize here some processes in which inhibition of Cdc42 may be a general mechanism to regulate polarized growth also under physiological conditions.This work is supported by grant PGC2018-093920-B-I00 to E.H. and PGC2018-097248-B-I00 to J.A., funded by MCIN/AEI/10.13039/501100011033 and by ‘European Regional Development Fund (ERDF) A way of making Europe’, by the ‘European Union’. The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Excellence Unit “María de Maeztu” Grant CEX2018-000792-M funded by MCIN/AEI/10.13039/501100011033. E.H. is a recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain). P.P. was supported by grants CSI150P20 and ‘Escalera de Excelencia’ CLU-2017-03 (Junta de Castilla y Leon, Spain; and the European Regional Development Fund, FEDER, EU). C.S-C. was a recipient of a María de Maeztu predoctoral fellowship from the Ministerio de Economía y Competitividad (Spain)

The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2

In response to hydrogen peroxide (H2O2), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H2O2 induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H2O2, but alternative effectors.Spanish Ministry of Science and Innovation (BFU2009-06933), PLAN E and FEDER, by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020, and by SGR2009-196 from Generalitat de Catalunya (Spain) to E.H; ICREA Academia Awards (Generalitat de Catalunya) to E. H. and J.A.; pre-doctoral fellowship (FPI) and a post-doctoral contract Juan de la Cierva, respectively, from the Ministerio de Ciencia e Innovación (Spain) to I.A.C. and P.G. Funding for this work and for open access charge: Spanish Ministry of Science and Innovatio