Ribosome biogenesis requires a vast number of transacting factors many of which are required for ... more Ribosome biogenesis requires a vast number of transacting factors many of which are required for the chemical modi®cation and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60±80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.
The use of interspecific hybrids during the industrial fermentation process has been well establi... more The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. We used hierarchical clustering heatmapping with principal component analysis for metabolic profiling and found that the low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic p...
The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects a... more The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects are yet to be deciphered completely. The additives in commercial formulations of glyphosate, though labeled as inert when used individually, have adverse effects when used in combination with other additives and the active ingredient. As a species,Saccharomyces cerevisiaehas a wide range of resistance to glyphosate-based herbicides. To investigate the underlying genetic differences between sensitive and resistant strains, global changes in gene expression were measured when yeast were exposed to a commercial formulation of glyphosate (CFG). Changes in gene expression involved in numerous pathways such as DNA replication, MAPK signaling, meiosis, and cell wall synthesis. Because so many diverse pathways were affected, these strains were then subjected to in-lab-evolutions (ILE) to select mutations that confer increased resistance. Common fragile sites were found to play a role in adaptatio...
The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK tha... more The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK that is conserved from yeast to humans and plays a central role in transcription. However, not all the components are required for basal transcription. Components of the tail are not essential but to varying degrees are required for growth in different stresses. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic, yet yeast can elicit a successful stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that a naturally occurring Med15 allele with polyQ (polyglutamine) expansion conferred MCHM sensitivity. Expansion in polyQ repeat can induce protein aggregation and in humans can cause neurodegenerative diseases. In yeast, the MCHM sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive and the homozyg...
On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and prop... more On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM’s toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. UsingS. cerevisiaeas a model organism we study the effect of acute exposition to crude MCHM on metabolism. Yeasts were treated with MCHM 3.9 mM in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metab...
While drugs and other industrial chemicals are routinely studied to assess risks, many widely-use... more While drugs and other industrial chemicals are routinely studied to assess risks, many widely-used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking-water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cell function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM and transcriptomics and mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to ...
Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex requ... more Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex required for all cell growth occurs through the association of supcomplexes in a stepwise manner.
The SSU processome is required for production of the small ribosomal subunit RNA, the 18S rRNA. S... more The SSU processome is required for production of the small ribosomal subunit RNA, the 18S rRNA. Specifically, the U3 small nucleolar RNA (snoRNA) component of the SSU processome is essential for the formation of the conserved central pseudoknot and for cleavages of the pre-rRNA, both of which are required for 18S maturation. To further elucidate how these events are mediated, we examined the regulatory and mechanistic roles of the U3 specific proteins: Imp3p, Imp4p, and Mpp10p. We found that these proteins demonstrated an interdependence with respect to their stability and to their association with the U3 snoRNA. Because mutations in the U3 snoRNA that disrupt pre-rRNA processing confer similar defects on growth and pre-rRNA processing as do carboxy-terminal truncations of Mpp10p, we hypothesized that Mpp10p may be involved in maintaining U3 snoRNA-pre-rRNA base pairing. However, combining the two mutations resulted in a more pronounced cleavage defect at site A 2 , suggesting that ...
Chemical spills and accidents contaminate the environment and disrupt societies and economies aro... more Chemical spills and accidents contaminate the environment and disrupt societies and economies around the globe. In the United States there were approximately 172,000 chemical spills that affected US waterbodies from 2004 to 2014. More than 8000 of these spills involved non-petroleum-related chemicals. Traditional emergency responses or incident command structures (ICSs) that respond to chemical spills require coordinated efforts by predominantly government personnel from multiple disciplines, including disaster management, public health, and environmental protection. However, the requirements of emergency response teams for science support might not be met within the traditional ICS. We describe the US ICS as an example of emergency-response approaches to chemical spills and provide examples in which external scientific support from research personnel benefitted the ICS emergency response, focusing primarily on nonpetroleum chemical spills. We then propose immediate, near-term, and ...
The characterization and public release of genome sequences from thousands of organisms is expand... more The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which ...
The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of th... more The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of the U3 snoRNA and at least 28 protein components, that is required for biogenesis of the 18S rRNA. We tested the function of one protein±protein interaction in the SSU processome, Mpp10p±Imp4p, in ribosome biogenesis. Exploiting the reverse twohybrid system, we screened for mutated Imp4 proteins that were conditionally defective for interaction with Mpp10p. Three different imp4 sequences were isolated that: (i) conferred conditional growth in the two-hybrid strain; (ii) complemented the disrupted imp4; (iii) conferred conditional growth in the context of their normal cellular function; and (iv) resulted in defective pre-rRNA processing at the non-permissive temperatures. Domain swapping revealed that mutations that conferred cold sensitivity resided in the N-terminal coiled-coil domain while mutations in the C-terminus conferred temperature sensitivity. Surprisingly, the mutated Imp4 proteins were not measurably defective for interaction with Mpp10p in the context of the SSU processome. This suggests that other members of the complex may contribute to maintaining the Mpp10p±Imp4p interaction in this large RNP. Since protein±protein interactions are critical for many different aspects of cellular metabolism, our work has implications for the study of other large protein complexes.
The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that resul... more The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5 0-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit.
mof6 - 1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promot... more mof6 - 1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed −1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6 - 1 is a unique allele of the histone deacetylase RPD3 , that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer per...
Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribos... more Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.
The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of th... more The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5′ end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins fi...
S. cerevisiae from different environments are subject to a wide range of selective pressures, whe... more S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resistant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to the transportation of glyphosate in and out of the cell. Taking advantage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells.
Background-Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare heredi... more Background-Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects. Objective-We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequence of 5 patients and their healthy direct relatives from 5 unrelated families. Methods-We performed whole exome sequencing on 5 CID-MIA patients and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene TTC7A on 3 additional CID-MIA patients. Results-Through analysis and comparison of the exomic sequence of the individuals from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in two of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from two patients with CID-MIA. Conclusions-We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA. Clinical Implications-Damaging mutations in the gene TTC7A should be scrutinized in patients with CID-MIA. Characterization of the role of this protein in the immune system and intestinal development, as well as in thymic epithelial cells may have important therapeutic implications.
The use of interspecific hybrids during the industrial fermentation process has been well establi... more The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. Hierarchical clustering heatmapping with principal component analysis for metabolic profiling was used in finding low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic profile of parental strains, while metabolic analysis suggested S. arboricola had a lower amino acid net uptake than S. cerevisiae. Additionally, this research found an increase in ethanolic fermentation from pyruvate and increased sulfur metabolism. Together, these results suggest S. arboricola is poised for in-depth metabolomic exploration in sake fermentation.
Chemical spills and accidents contaminate the environment and disrupt societies and economies aro... more Chemical spills and accidents contaminate the environment and disrupt societies and economies around the globe. In the United States there were approximately 172,000 chemical spills that affected US waterbodies from 2004 to 2014. More than 8000 of these spills involved non-petroleum-related chemicals. Traditional emergency responses or incident command structures (ICSs) that respond to chemical spills require coordinated efforts by predominantly government personnel from multiple disciplines, including disaster management, public health, and environmental protection. However, the requirements of emergency response teams for science support might not be met within the traditional ICS. We describe the US ICS as an example of emergency-response approaches to chemical spills and provide examples in which external scientific support from research personnel benefitted the ICS emergency response, focusing primarily on nonpetroleum chemical spills. We then propose immediate, near-term, and long-term activities to support the response to chemical spills, focusing on nonpetroleum chemical spills. Further, we call for science support for spill prevention and near-term spill-incident response and identify longer-term research needs. The development of a formal mechanism for external science support of ICS from governmental and nongovernmental scientists would benefit rapid responders, advance incident-and crisis-response science, and aid society in coping with and recovering from chemical spills.
One sentence summary: Cell growth requires the production of mature ribosomes and the assembly pr... more One sentence summary: Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex required for all cell growth occurs through the association of supcomplexes in a stepwise manner. ABSTRACT Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling highly structured rRNAs with ribosomal proteins. Transcription and processing of pre-rRNAs are linked by the transcription-Utp sub-complex (t-Utps), a sub-complex of the small subunit (SSU) processome and prompted the investigations for the requirements of t-Utp formation and transition into the SSU processome. The rDNA promoter, the first 44 nucleotides of the 5 ETS, and active transcription by pol I were sufficient to recruit the t-Utps to the rDNA. Pol5, accessory factor, dissociated as t-Utps matured into the UtpA complex which permitted later recruitment of the UtpB, U3 snoRNP and the Mpp10 complex into the SSU processome. The t-Utp complex associated with short RNAs 121 and 138 nucleotides long transcribed from the 5 ETS. These transcripts were not present when pol II transcribed the rDNA or in nondividing cells. Depletion of a t-Utp, but not of other SSU processome components led to decreased levels of the short transcripts. However, ectopic expression of the short transcripts slowed the growth of yeast with impaired rDNA transcription. These results provide insight into how transcription of the rRNA primes the assemble of t-Utp complex with the pre-rRNA into the UtpA complex and the later association of SSU processome components.
Ribosome biogenesis requires a vast number of transacting factors many of which are required for ... more Ribosome biogenesis requires a vast number of transacting factors many of which are required for the chemical modi®cation and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60±80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.
The use of interspecific hybrids during the industrial fermentation process has been well establi... more The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. We used hierarchical clustering heatmapping with principal component analysis for metabolic profiling and found that the low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic p...
The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects a... more The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects are yet to be deciphered completely. The additives in commercial formulations of glyphosate, though labeled as inert when used individually, have adverse effects when used in combination with other additives and the active ingredient. As a species,Saccharomyces cerevisiaehas a wide range of resistance to glyphosate-based herbicides. To investigate the underlying genetic differences between sensitive and resistant strains, global changes in gene expression were measured when yeast were exposed to a commercial formulation of glyphosate (CFG). Changes in gene expression involved in numerous pathways such as DNA replication, MAPK signaling, meiosis, and cell wall synthesis. Because so many diverse pathways were affected, these strains were then subjected to in-lab-evolutions (ILE) to select mutations that confer increased resistance. Common fragile sites were found to play a role in adaptatio...
The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK tha... more The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK that is conserved from yeast to humans and plays a central role in transcription. However, not all the components are required for basal transcription. Components of the tail are not essential but to varying degrees are required for growth in different stresses. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic, yet yeast can elicit a successful stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that a naturally occurring Med15 allele with polyQ (polyglutamine) expansion conferred MCHM sensitivity. Expansion in polyQ repeat can induce protein aggregation and in humans can cause neurodegenerative diseases. In yeast, the MCHM sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive and the homozyg...
On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and prop... more On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM’s toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. UsingS. cerevisiaeas a model organism we study the effect of acute exposition to crude MCHM on metabolism. Yeasts were treated with MCHM 3.9 mM in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metab...
While drugs and other industrial chemicals are routinely studied to assess risks, many widely-use... more While drugs and other industrial chemicals are routinely studied to assess risks, many widely-used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking-water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cell function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM and transcriptomics and mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to ...
Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex requ... more Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex required for all cell growth occurs through the association of supcomplexes in a stepwise manner.
The SSU processome is required for production of the small ribosomal subunit RNA, the 18S rRNA. S... more The SSU processome is required for production of the small ribosomal subunit RNA, the 18S rRNA. Specifically, the U3 small nucleolar RNA (snoRNA) component of the SSU processome is essential for the formation of the conserved central pseudoknot and for cleavages of the pre-rRNA, both of which are required for 18S maturation. To further elucidate how these events are mediated, we examined the regulatory and mechanistic roles of the U3 specific proteins: Imp3p, Imp4p, and Mpp10p. We found that these proteins demonstrated an interdependence with respect to their stability and to their association with the U3 snoRNA. Because mutations in the U3 snoRNA that disrupt pre-rRNA processing confer similar defects on growth and pre-rRNA processing as do carboxy-terminal truncations of Mpp10p, we hypothesized that Mpp10p may be involved in maintaining U3 snoRNA-pre-rRNA base pairing. However, combining the two mutations resulted in a more pronounced cleavage defect at site A 2 , suggesting that ...
Chemical spills and accidents contaminate the environment and disrupt societies and economies aro... more Chemical spills and accidents contaminate the environment and disrupt societies and economies around the globe. In the United States there were approximately 172,000 chemical spills that affected US waterbodies from 2004 to 2014. More than 8000 of these spills involved non-petroleum-related chemicals. Traditional emergency responses or incident command structures (ICSs) that respond to chemical spills require coordinated efforts by predominantly government personnel from multiple disciplines, including disaster management, public health, and environmental protection. However, the requirements of emergency response teams for science support might not be met within the traditional ICS. We describe the US ICS as an example of emergency-response approaches to chemical spills and provide examples in which external scientific support from research personnel benefitted the ICS emergency response, focusing primarily on nonpetroleum chemical spills. We then propose immediate, near-term, and ...
The characterization and public release of genome sequences from thousands of organisms is expand... more The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which ...
The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of th... more The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of the U3 snoRNA and at least 28 protein components, that is required for biogenesis of the 18S rRNA. We tested the function of one protein±protein interaction in the SSU processome, Mpp10p±Imp4p, in ribosome biogenesis. Exploiting the reverse twohybrid system, we screened for mutated Imp4 proteins that were conditionally defective for interaction with Mpp10p. Three different imp4 sequences were isolated that: (i) conferred conditional growth in the two-hybrid strain; (ii) complemented the disrupted imp4; (iii) conferred conditional growth in the context of their normal cellular function; and (iv) resulted in defective pre-rRNA processing at the non-permissive temperatures. Domain swapping revealed that mutations that conferred cold sensitivity resided in the N-terminal coiled-coil domain while mutations in the C-terminus conferred temperature sensitivity. Surprisingly, the mutated Imp4 proteins were not measurably defective for interaction with Mpp10p in the context of the SSU processome. This suggests that other members of the complex may contribute to maintaining the Mpp10p±Imp4p interaction in this large RNP. Since protein±protein interactions are critical for many different aspects of cellular metabolism, our work has implications for the study of other large protein complexes.
The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that resul... more The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5 0-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit.
mof6 - 1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promot... more mof6 - 1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed −1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6 - 1 is a unique allele of the histone deacetylase RPD3 , that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer per...
Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribos... more Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.
The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of th... more The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5′ end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins fi...
S. cerevisiae from different environments are subject to a wide range of selective pressures, whe... more S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resistant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to the transportation of glyphosate in and out of the cell. Taking advantage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells.
Background-Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare heredi... more Background-Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects. Objective-We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequence of 5 patients and their healthy direct relatives from 5 unrelated families. Methods-We performed whole exome sequencing on 5 CID-MIA patients and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene TTC7A on 3 additional CID-MIA patients. Results-Through analysis and comparison of the exomic sequence of the individuals from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in two of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from two patients with CID-MIA. Conclusions-We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA. Clinical Implications-Damaging mutations in the gene TTC7A should be scrutinized in patients with CID-MIA. Characterization of the role of this protein in the immune system and intestinal development, as well as in thymic epithelial cells may have important therapeutic implications.
The use of interspecific hybrids during the industrial fermentation process has been well establi... more The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. Hierarchical clustering heatmapping with principal component analysis for metabolic profiling was used in finding low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic profile of parental strains, while metabolic analysis suggested S. arboricola had a lower amino acid net uptake than S. cerevisiae. Additionally, this research found an increase in ethanolic fermentation from pyruvate and increased sulfur metabolism. Together, these results suggest S. arboricola is poised for in-depth metabolomic exploration in sake fermentation.
Chemical spills and accidents contaminate the environment and disrupt societies and economies aro... more Chemical spills and accidents contaminate the environment and disrupt societies and economies around the globe. In the United States there were approximately 172,000 chemical spills that affected US waterbodies from 2004 to 2014. More than 8000 of these spills involved non-petroleum-related chemicals. Traditional emergency responses or incident command structures (ICSs) that respond to chemical spills require coordinated efforts by predominantly government personnel from multiple disciplines, including disaster management, public health, and environmental protection. However, the requirements of emergency response teams for science support might not be met within the traditional ICS. We describe the US ICS as an example of emergency-response approaches to chemical spills and provide examples in which external scientific support from research personnel benefitted the ICS emergency response, focusing primarily on nonpetroleum chemical spills. We then propose immediate, near-term, and long-term activities to support the response to chemical spills, focusing on nonpetroleum chemical spills. Further, we call for science support for spill prevention and near-term spill-incident response and identify longer-term research needs. The development of a formal mechanism for external science support of ICS from governmental and nongovernmental scientists would benefit rapid responders, advance incident-and crisis-response science, and aid society in coping with and recovering from chemical spills.
One sentence summary: Cell growth requires the production of mature ribosomes and the assembly pr... more One sentence summary: Cell growth requires the production of mature ribosomes and the assembly protein-RNA complex required for all cell growth occurs through the association of supcomplexes in a stepwise manner. ABSTRACT Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling highly structured rRNAs with ribosomal proteins. Transcription and processing of pre-rRNAs are linked by the transcription-Utp sub-complex (t-Utps), a sub-complex of the small subunit (SSU) processome and prompted the investigations for the requirements of t-Utp formation and transition into the SSU processome. The rDNA promoter, the first 44 nucleotides of the 5 ETS, and active transcription by pol I were sufficient to recruit the t-Utps to the rDNA. Pol5, accessory factor, dissociated as t-Utps matured into the UtpA complex which permitted later recruitment of the UtpB, U3 snoRNP and the Mpp10 complex into the SSU processome. The t-Utp complex associated with short RNAs 121 and 138 nucleotides long transcribed from the 5 ETS. These transcripts were not present when pol II transcribed the rDNA or in nondividing cells. Depletion of a t-Utp, but not of other SSU processome components led to decreased levels of the short transcripts. However, ectopic expression of the short transcripts slowed the growth of yeast with impaired rDNA transcription. These results provide insight into how transcription of the rRNA primes the assemble of t-Utp complex with the pre-rRNA into the UtpA complex and the later association of SSU processome components.
On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and prop... more On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM's toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. Using S. cerevisiae as a model organism we study the effect of acute exposition to crude MCHM on metabolism. Yeasts were treated with MCHM 3.9 mM in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metabolic network models (GSMNM) of MCHM treated vs non-treated control. We integrated the effect of MCHM on yeast gene expression from RNA-Seq data within these GSMNM. 181 and 66 metabolites were identified by the ESI-MS and GC-MS procedures, respectively. From these 38 and 34 relevant metabolites were selected from ESI-MS and GC-MS respectively, for 72 unique compounds. MCHM induced amino acid accumulation, via its effects on amino acid metabolism, as well as a potential impairment of ribosome biogenesis. MCHM affects phospholipid biosynthesis and decrease the levels of ergosterol, with a potential impact in the biophysical properties of yeast cellular membranes. The FBA simulations were able to reproduce the deleterious effect of MCHM on cell's growth and suggest that the effect of MCHM on ubiquinol:ferricytochrome c reductase reaction, caused by the under-expression of CYT1 gene, could be the driven force behind the observed effect on yeast metabolism and growth.
While drugs and other industrial chemicals are routinely studied to assess risks, many widely use... more While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM, transcriptomics and, mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, which is involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM, and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein interactions, while zinc induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM.
S. cerevisiae from different environments are subject to a wide range of selective pressures, whe... more S. cerevisiae from different environments are subject to a wide range of selective pressures, whether intentional or by happenstance. Chemicals classified by their application, such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Crops engineered to be resistant to glyphosate contain a homolog of EPSPS that is not bound by glyphosate. Here, we show that S. cerevisiae has a wide-range of glyphosate resistance. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in an ABC multiple drug transporter, Pdr5, and an amino acid permease, Dip5. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to the transportation of glyphosate in and out of the cell. Taking advantage of the natural genetic variation within yeast and measuring growth under different conditions that would change the use of the shikimate pathway, we uncovered a general transport mechanism of glyphosate into eukaryotic cells.
The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK tha... more The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK that is conserved from yeast to humans and plays a central role in transcription. However, not all the components are required for basal transcription. Components of the tail are not essential but to varying degrees are required for growth in different stresses. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic, yet yeast can elicit a successful stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that a naturally occurring Med15 allele with polyQ (polyglutamine) expansion conferred MCHM sensitivity. Expansion in polyQ repeat can induce protein aggregation and in humans can cause neurodegenerative diseases. In yeast, the MCHM sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive and the homozygous null mutant was less sensitive than the hemizygous hybrids. This suggests that there is an incompatibility between Mediator components from genetic divergent yeast strains. Transcriptomics from yeast expressing the incompatible Med15 (longer polyQ repeats in the strain with fewer repeats) changed gene expression in diverse pathways. Med15 protein existed in multiple isoforms, mostly from likely post-translational modifications and different alleles have different patterns of isoforms. Stability of both alleles of Med15 was dependent on Ydj1, a J-type chaperone. The protein level of the incompatible Med15 allele was lower than the compatible allele and was turned over faster. Med15 is tethered to the rest of the Mediator complex via Med2 and 3. Deletion of either Med2 or Med3 changed the Med15 isoform patterns in a similar manner. Whereas deletion of Med5, a distal component of the Mediator tail, did not change the pattern. The med2 and med3 mutants were similarly sensitive to MCHM while med5 mutants were not. Differences in the phenotype of yeast carrying different Med15 alleles extend to other stresses. The incompatible allele of Med15 improved growth of yeast to chemicals that produce free radicals and the compatible allele of Med15 improved growth to reducing agents, caffeine, and hydroxyurea. Med15 directly interacts with Gcn4 and other TFs and in vitro form phase-separated droplets. This variation may reflect the positive and negative role that Med15 has in transcription. Genetic variation in transcriptional regulators can magnify differences in response to environmental changes, in contrast, genetic variation in a metabolic enzyme. These polymorphic control genes are master variators.
The characterization and public release of genome sequences from thousands of organisms is expand... more The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community.
The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects a... more The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects are yet to be deciphered completely. The additives in commercial formulations of glyphosate, though labeled as inert when used individually, have adverse effects when used in combination with other additives and the active ingredient. As a species, Saccharomyces cerevisiae has a wide range of resistance to glyphosate-based herbicides. To investigate the underlying genetic differences between sensitive and resistant strains, global changes in gene expression were measured when yeast were exposed to a commercial formulation of glyphosate (CFG). Changes in gene expression involved in numerous pathways such as DNA replication, MAPK signaling, meiosis, and cell wall synthesis. Because so many diverse pathways were affected, these strains were then subjected to in-lab-evolutions (ILE) to select mutations that confer increased resistance. Common fragile sites were found to play a role in adaptation mechanisms used by cells to attain resistance with long-term exposure to CFG. The cell wall structure acts as a protective barrier in alleviating the stress caused by exposure to CFG. The thicker the cell wall, the more resistant the cell is against CFG. Hence, a detailed study of the changes occurring at the genome and transcriptome level is essential to better understand the possible effects of CFG on the cell as a whole.
Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling h... more Ribosomes are synthesized by large ribonucleoprotein complexes cleaving and properly assembling highly structured rRNAs with ribosomal proteins. Transcription and processing of pre-rRNAs are linked by the transcription-Utp sub-complex (t-Utps), a sub-complex of the small subunit (SSU) processome and prompted the investigations for the requirements of t-Utp formation and transition into the SSU processome. The rDNA promoter, the first 44 nucleotides of the 5΄ETS, and active transcription by pol I were sufficient to recruit the t-Utps to the rDNA. Pol5, accessory factor, dissociated as t-Utps matured into the UtpA complex which permitted later recruitment of the UtpB, U3 snoRNP and the Mpp10 complex into the SSU processome. The t-Utp complex associated with short RNAs 121 and 138 nucleotides long transcribed from the 5΄ETS. These transcripts were not present when pol II transcribed the rDNA or in nondividing cells. Depletion of a t-Utp, but not of other SSU processome components led to decreased levels of the short transcripts. However, ectopic expression of the short transcripts slowed the growth of yeast with impaired rDNA transcription. These results provide insight into how transcription of the rRNA primes the assemble of t-Utp complex with the pre-rRNA into the UtpA complex and the later association of SSU processome components.
Cellular metabolism can change the potency of a chemical's tumorigenicity. 4-nitroquinoline-1-oxi... more Cellular metabolism can change the potency of a chemical's tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO inSaccharomyces cerevisiaeTo study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenicScerevisiaestrains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression ofSNQ2(a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression ofSNQ2alone was sufficient to overcome 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci includingSNQ2In addition to a known Yrr1 targetSNG1, Yrr1 also bound upstream ofRPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including thede novopurine pathway, and the ribonuclease reductase pathways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of theADEgenes in thede novopurine biosynthetic pathway, the mutant Yrr1 increased expression ofADEgenes even in the absence of 4NQO. These sameADEgenes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization.
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