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Gregor Hogenauer - One of the best experts on this subject based on the ideXlab platform.
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Diazaborine treatment of yeast cells inhibits maturation of the 60s ribosomal subunit
Molecular and Cellular Biology, 2004Co-Authors: Brigitte Pertschy, Gertrude Zisser, Hermine Schein, Rene Koffel, Gernot Rauch, Karlheinz Grillitsch, Christina Morgenstern, Michael Durchschlag, Gregor Hogenauer, Helmut BerglerAbstract:Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after Diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/ Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that Diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of Diazaborine on rRNA processing. Translation of mRNA in the cytoplasm relies on the ribosome. While the principal steps in initiation and elongation of protein synthesis were uncovered in the middle of the last century, details of the biogenesis of the ribosome itself remained elusive. However, the development of novel techniques in affinity purification and refined techniques of protein identification by mass spectrometry within the last several years provided novel insights into the steps of assembly of the ribosomal subunits and how they are coordinated with the processing of the rRNA precursors (for recent reviews see references 15 and 47). Much of this detailed knowledge was worked out in the baker’s yeast, Saccharomyces cerevisiae. This organism constitutes a simple model for the reaction sequence occurring in more complex eukaryotic cells.
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the transporters pdr5p and snq2p mediate Diazaborine resistance and are under the control of the gain of function allele pdr1 12
FEBS Journal, 2004Co-Authors: Eva Wehrschutzsigl, Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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The transporters Pdr5p and Snq2p mediate Diazaborine resistance and are under the control of the gain‐of‐function allele PDR1‐12
European journal of biochemistry, 2004Co-Authors: Eva Wehrschütz‐sigl, Helmut Bergler, Helmut Jungwirth, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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Diazaborine Treatment of Baker's Yeast Results in Stabilization of Aberrant mRNAs
The Journal of biological chemistry, 2001Co-Authors: Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:Abstract Upon Northern blotting, Saccharomyces cerevisiae that was treated with Diazaborine showed aberrant mRNAs that were extended at the 3′-end and terminated at secondary processing sites. These bands were also detected in untreated Δupf1, Δxrn1, and rat7-1mutants. This finding demonstrates that the aberrant mRNAs also occur in untreated strains in small quantities and can reach the cytoplasm, where they are normally degraded by Xrn1p. Diazaborine treatment stabilizes these mRNAs. The detection of the aberrant bands in the untreated rat7-1 strain indicates that Rat7 is involved in quality control of RNA. The aberrant mRNAs were not detected after Diazaborine treatment of a DRG1-1mutant. Drg1p, a member of the family of AAA (ATPases associated with a variety of cellular activities) proteins, which are thought to represent specific chaperones, may be involved in the process of unfolding the mRNA-ribonucleoprotein complex or in the recognition of aberrant mRNA molecules in the cytoplasm.
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Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain-of-function allele of gene YAP1.
European journal of biochemistry, 2000Co-Authors: Helmut Jungwirth, Helmut Bergler, Franz Wendler, Barbara Platzer, Gregor HogenauerAbstract:We have found that YAP1-mediated Diazaborine resistance in the yeast Saccharomyces cerevisiae requires two efflux pumps, i.e. the major-facilitator-superfamily transporter Flr1p, which is located in the cytoplasmic membrane and the ATP-binding-cassette transporter Ycf1p which is present in the vacuolar membrane. Both these transporters are known to be under the control of the transcriptional transactivator Yap1p which explains our earlier finding that overexpression of YAP1 mediates Diazaborine resistance. Overexpression of YAP1 in a Δflr1Δycf1 double disruptant strain does not mediate any Diazaborine resistance, showing that these pumps are the only ones involved in detoxification of this drug. We also found a new mechanism of Diazaborine resistance which is caused by an allelic form of YAP1, designated YAP1-11. This allele of YAP1 carries a mutation that leads to a C620F exchange in the C-terminal cysteine-rich-domain region and is the first mutant of YAP1 that was isolated by a conventional genetic screen for drug resistance. The protein encoded by the gain-of-function allele may transactivate by a different mechanism from the wild-type protein when overexpressed because it does not enhance YCF1 mRNA and still mediates Diazaborine resistance in a Δflr1Δycf1 background.
Helmut Bergler - One of the best experts on this subject based on the ideXlab platform.
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The drug Diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1.
The Journal of biological chemistry, 2013Co-Authors: Mathias Loibl, Brigitte Pertschy, Gertrude Zisser, Isabella Klein, Michael Prattes, Claudia Schmidt, Lisa Kappel, Anna Gungl, Elmar Krieger, Helmut BerglerAbstract:The drug Diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of Diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.
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Diazaborine treatment of yeast cells inhibits maturation of the 60s ribosomal subunit
Molecular and Cellular Biology, 2004Co-Authors: Brigitte Pertschy, Gertrude Zisser, Hermine Schein, Rene Koffel, Gernot Rauch, Karlheinz Grillitsch, Christina Morgenstern, Michael Durchschlag, Gregor Hogenauer, Helmut BerglerAbstract:Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after Diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/ Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that Diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of Diazaborine on rRNA processing. Translation of mRNA in the cytoplasm relies on the ribosome. While the principal steps in initiation and elongation of protein synthesis were uncovered in the middle of the last century, details of the biogenesis of the ribosome itself remained elusive. However, the development of novel techniques in affinity purification and refined techniques of protein identification by mass spectrometry within the last several years provided novel insights into the steps of assembly of the ribosomal subunits and how they are coordinated with the processing of the rRNA precursors (for recent reviews see references 15 and 47). Much of this detailed knowledge was worked out in the baker’s yeast, Saccharomyces cerevisiae. This organism constitutes a simple model for the reaction sequence occurring in more complex eukaryotic cells.
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the transporters pdr5p and snq2p mediate Diazaborine resistance and are under the control of the gain of function allele pdr1 12
FEBS Journal, 2004Co-Authors: Eva Wehrschutzsigl, Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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The transporters Pdr5p and Snq2p mediate Diazaborine resistance and are under the control of the gain‐of‐function allele PDR1‐12
European journal of biochemistry, 2004Co-Authors: Eva Wehrschütz‐sigl, Helmut Bergler, Helmut Jungwirth, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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Diazaborine Treatment of Baker's Yeast Results in Stabilization of Aberrant mRNAs
The Journal of biological chemistry, 2001Co-Authors: Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:Abstract Upon Northern blotting, Saccharomyces cerevisiae that was treated with Diazaborine showed aberrant mRNAs that were extended at the 3′-end and terminated at secondary processing sites. These bands were also detected in untreated Δupf1, Δxrn1, and rat7-1mutants. This finding demonstrates that the aberrant mRNAs also occur in untreated strains in small quantities and can reach the cytoplasm, where they are normally degraded by Xrn1p. Diazaborine treatment stabilizes these mRNAs. The detection of the aberrant bands in the untreated rat7-1 strain indicates that Rat7 is involved in quality control of RNA. The aberrant mRNAs were not detected after Diazaborine treatment of a DRG1-1mutant. Drg1p, a member of the family of AAA (ATPases associated with a variety of cellular activities) proteins, which are thought to represent specific chaperones, may be involved in the process of unfolding the mRNA-ribonucleoprotein complex or in the recognition of aberrant mRNA molecules in the cytoplasm.
Helmut Jungwirth - One of the best experts on this subject based on the ideXlab platform.
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the transporters pdr5p and snq2p mediate Diazaborine resistance and are under the control of the gain of function allele pdr1 12
FEBS Journal, 2004Co-Authors: Eva Wehrschutzsigl, Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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The transporters Pdr5p and Snq2p mediate Diazaborine resistance and are under the control of the gain‐of‐function allele PDR1‐12
European journal of biochemistry, 2004Co-Authors: Eva Wehrschütz‐sigl, Helmut Bergler, Helmut Jungwirth, Gregor HogenauerAbstract:The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to Diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in Diazaborine detoxification. We report that in the PDR3-33 mutant Diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in Diazaborine detoxification. In addition, we show that in the presence of cycloheximide or Diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p.
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Diazaborine Treatment of Baker's Yeast Results in Stabilization of Aberrant mRNAs
The Journal of biological chemistry, 2001Co-Authors: Helmut Jungwirth, Helmut Bergler, Gregor HogenauerAbstract:Abstract Upon Northern blotting, Saccharomyces cerevisiae that was treated with Diazaborine showed aberrant mRNAs that were extended at the 3′-end and terminated at secondary processing sites. These bands were also detected in untreated Δupf1, Δxrn1, and rat7-1mutants. This finding demonstrates that the aberrant mRNAs also occur in untreated strains in small quantities and can reach the cytoplasm, where they are normally degraded by Xrn1p. Diazaborine treatment stabilizes these mRNAs. The detection of the aberrant bands in the untreated rat7-1 strain indicates that Rat7 is involved in quality control of RNA. The aberrant mRNAs were not detected after Diazaborine treatment of a DRG1-1mutant. Drg1p, a member of the family of AAA (ATPases associated with a variety of cellular activities) proteins, which are thought to represent specific chaperones, may be involved in the process of unfolding the mRNA-ribonucleoprotein complex or in the recognition of aberrant mRNA molecules in the cytoplasm.
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Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain-of-function allele of gene YAP1.
European journal of biochemistry, 2000Co-Authors: Helmut Jungwirth, Helmut Bergler, Franz Wendler, Barbara Platzer, Gregor HogenauerAbstract:We have found that YAP1-mediated Diazaborine resistance in the yeast Saccharomyces cerevisiae requires two efflux pumps, i.e. the major-facilitator-superfamily transporter Flr1p, which is located in the cytoplasmic membrane and the ATP-binding-cassette transporter Ycf1p which is present in the vacuolar membrane. Both these transporters are known to be under the control of the transcriptional transactivator Yap1p which explains our earlier finding that overexpression of YAP1 mediates Diazaborine resistance. Overexpression of YAP1 in a Δflr1Δycf1 double disruptant strain does not mediate any Diazaborine resistance, showing that these pumps are the only ones involved in detoxification of this drug. We also found a new mechanism of Diazaborine resistance which is caused by an allelic form of YAP1, designated YAP1-11. This allele of YAP1 carries a mutation that leads to a C620F exchange in the C-terminal cysteine-rich-domain region and is the first mutant of YAP1 that was isolated by a conventional genetic screen for drug resistance. The protein encoded by the gain-of-function allele may transactivate by a different mechanism from the wild-type protein when overexpressed because it does not enhance YCF1 mRNA and still mediates Diazaborine resistance in a Δflr1Δycf1 background.
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Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.
The Journal of biological chemistry, 1997Co-Authors: Franz Wendler, Gertrude Zisser, Helmut Bergler, Helmut Jungwirth, Katja Prutej, Karl Kuchler, Gregor HogenauerAbstract:Abstract We have investigated the mechanisms underlying resistance to the drug Diazaborine in Saccharomyces cerevisiae. We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups. The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, andPDR3. The AFG2 gene encodes an AAA (ATPases associated to a variety of cellularactivities) protein of unknown function, whilePDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development. The isolated PDR1–12 and PDR3–33alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively. In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a Diazaborine-resistant phenotype. The YAP1-mediated Diazaborine resistance is dependent on the presence of functionalPDR1 and PDR3 genes, although PDR3had a more pronounced effect. These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.
Gertrude Zisser - One of the best experts on this subject based on the ideXlab platform.
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Viewing pre-60S maturation at a minute's timescale.
Nucleic acids research, 2017Co-Authors: Gertrude Zisser, Brigitte Pertschy, Isabella Klein, Michael Prattes, Uli Ohmayer, Christina Mauerhofer, Valentin Mitterer, Gerald N. Rechberger, Heimo Wolinski, Philipp MilkereitAbstract:The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor Diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by Diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation.
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The drug Diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1.
The Journal of biological chemistry, 2013Co-Authors: Mathias Loibl, Brigitte Pertschy, Gertrude Zisser, Isabella Klein, Michael Prattes, Claudia Schmidt, Lisa Kappel, Anna Gungl, Elmar Krieger, Helmut BerglerAbstract:The drug Diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of Diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.
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Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1
Molecular and Cellular Biology, 2007Co-Authors: Brigitte Pertschy, Gertrude Zisser, Lisa Kappel, Cosmin Saveanu, Alice Lebreton, Martin Tengg, Alain Jacquier, Eva Liebminger, Berthold Nobis, Ida Van Der KleiAbstract:Allelic forms of DRG1/AFG2 confer resistance to the drug Diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.
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Diazaborine treatment of yeast cells inhibits maturation of the 60s ribosomal subunit
Molecular and Cellular Biology, 2004Co-Authors: Brigitte Pertschy, Gertrude Zisser, Hermine Schein, Rene Koffel, Gernot Rauch, Karlheinz Grillitsch, Christina Morgenstern, Michael Durchschlag, Gregor Hogenauer, Helmut BerglerAbstract:Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after Diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/ Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that Diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of Diazaborine on rRNA processing. Translation of mRNA in the cytoplasm relies on the ribosome. While the principal steps in initiation and elongation of protein synthesis were uncovered in the middle of the last century, details of the biogenesis of the ribosome itself remained elusive. However, the development of novel techniques in affinity purification and refined techniques of protein identification by mass spectrometry within the last several years provided novel insights into the steps of assembly of the ribosomal subunits and how they are coordinated with the processing of the rRNA precursors (for recent reviews see references 15 and 47). Much of this detailed knowledge was worked out in the baker’s yeast, Saccharomyces cerevisiae. This organism constitutes a simple model for the reaction sequence occurring in more complex eukaryotic cells.
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Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.
The Journal of biological chemistry, 1997Co-Authors: Franz Wendler, Gertrude Zisser, Helmut Bergler, Helmut Jungwirth, Katja Prutej, Karl Kuchler, Gregor HogenauerAbstract:Abstract We have investigated the mechanisms underlying resistance to the drug Diazaborine in Saccharomyces cerevisiae. We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups. The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, andPDR3. The AFG2 gene encodes an AAA (ATPases associated to a variety of cellularactivities) protein of unknown function, whilePDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development. The isolated PDR1–12 and PDR3–33alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively. In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a Diazaborine-resistant phenotype. The YAP1-mediated Diazaborine resistance is dependent on the presence of functionalPDR1 and PDR3 genes, although PDR3had a more pronounced effect. These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.
Matthew S. Ward - One of the best experts on this subject based on the ideXlab platform.
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Identification of cellular targets of a series of boron heterocycles using TIPA II—A sensitive target identification platform
Bioorganic & medicinal chemistry, 2016Co-Authors: Matthew S. Ward, Divya Kanichar, Lance Roppiyakuda, Ewa Kosmowska, Isba Silva, Walfre Martinez, Jameka Jefferson, Shakila Rahman, Jeanie M. Garcia, Michelle FaustAbstract:One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, Diazaborine and thiolactomycin, further validating the utility of the new system.
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A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds
Applied Microbiology and Biotechnology, 2014Co-Authors: Isba Silva, Lilian J. Real, Matthew S. WardAbstract:One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli . From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, Diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.