RESUMO
The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.
Assuntos
Regiões 3' não Traduzidas , Transportador de Cobre 1 , Cobre , Regulação Fúngica da Expressão Gênica , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Nucleares , RNA Mensageiro , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cobre/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Códon sem Sentido/genéticaRESUMO
The highly conserved Nonsense-mediated mRNA decay (NMD) pathway is a translation dependent mRNA degradation pathway. Although NMD is best known for its role in degrading mRNAs with premature termination codons (PTCs) generated during transcription, splicing, or damage to the mRNAs, NMD is now also recognized as a pathway with additional important functions. Notably, NMD precisely regulates protein coding natural mRNAs, hence controlling gene expression within several physiologically significant pathways. Such pathways affected by NMD include nutritional bio-metal homeostasis and metal ion detoxification, as well as crosstalk between these pathways. Here, we focus on the relationships between NMD and various metal homeostasis and detoxification pathways. We review the described role that the NMD pathway plays in magnesium, zinc, iron, and copper homeostasis, as well as cadmium detoxification.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , RNA Mensageiro/metabolismo , Homeostase/genéticaRESUMO
Regulation of mRNA steady-state levels is important in controlling gene expression particularly in response to environmental stimuli. This allows cells to rapidly respond to environment changes. The highly conserved nonsense-mediated mRNA decay (NMD) pathway was initially identified as a pathway that degrades aberrant mRNAs. NMD is now recognized as a pathway with additional functions including precisely regulating the expression of select natural mRNAs. Majority of these natural mRNAs encode fully functional proteins. Regulation of natural mRNAs by NMD is activated by NMD targeting features and environmental cues. Here, we show that Saccharomyces cerevisiae strains from three genetic backgrounds respond differentially to NMD depending on the environmental stimuli. We found that wild type and NMD mutant W303a, BY4741, and RM11-1a yeast strains respond similarly to copper in the environment but respond differentially to toxic cadmium. Furthermore, the PCA1 alleles encoding different mRNAs from W303a and RM11-1a strains are regulated similarly by NMD in response to the bio-metal copper but differentially in response to toxic cadmium.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Fúngica da Expressão Gênica , Patrimônio Genético , Íons , Degradação do RNAm Mediada por Códon sem Sentido , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The differential regulation of COX17, COX19 and COX23 mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was investigated. The NMD pathway regulates mRNAs that aberrantly terminate translation. This includes mRNAs harboring premature translation termination codons and natural mRNAs. Most natural mRNAs regulated by NMD encode fully functional proteins involved in various cellular processes. However, the cause and targeting of most of these mRNAs by the pathway is not understood. Analysis of a set of mRNAs involved in copper homeostasis showed that a subset of these mRNAs function in mitochondrial copper homeostasis. Here, we examined the regulation of COX17, COX19 and COX23 mRNAs by NMD. These mRNAs encode homologous mitochondrial proteins involved in metallation of cytochrome c oxidase. We found that COX17, COX19 and COX23 mRNAs are differentially regulated by NMD depending on environmental copper levels. A long 3'-UTR contributes to the direct regulation of COX19 mRNA by the pathway. Alternatively, COX23 mRNA contains a long 3'-UTR, but is indirectly regulated by the pathway under two conditions tested here. Analysis of the functionality of the NMD targeting features in COX23 mRNA showed that the COX23 3'-UTR is sufficient to trigger NMD. The regulation of mRNAs involved in mitochondrial copper metabolism by NMD is physiologically significant because excess copper enhances growth of NMD mutants on a non-fermentable carbon source. These findings suggest that regulation of mRNAs encoding homologous proteins by NMD can be differential depending on environmental copper levels. Furthermore, these findings suggest copper ion homeostatic mechanisms in the mitochondria occur at the mRNA level via the NMD pathway.
Assuntos
Proteínas de Transporte de Cátions/genética , Regulação Fúngica da Expressão Gênica , Chaperonas Moleculares/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Cobre/farmacologia , Proteínas de Transporte de Cobre , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The nonsense-mediated mRNA decay pathway (NMD) is an mRNA degradation pathway that degrades mRNAs that prematurely terminate translation. These mRNAs include mRNAs with premature termination codons as well as many natural mRNAs. In Saccharomyces cerevisiae a number of features have been shown to target natural mRNAs to NMD. However, the extent to which natural mRNAs from the same functional group are regulated by NMD and how environmental conditions influence this regulation is not known. Here, we examined mRNAs involved in copper homeostasis and are predicted to be sensitive to NMD. We found that the majority of these mRNAs have long 3'-UTRs that could target them for degradation by NMD. Analysis of one of these mRNAs, COX19, found that the long 3'-UTR contributes to regulation of this mRNA by NMD. Furthermore, we examined an additional mRNA, MAC1 under low copper conditions. We found that low copper growth conditions affect NMD sensitivity of the MAC1 mRNA demonstrating that sensitivity to NMD can be altered by environmental conditions. MAC1 is a copper sensitive transcription factor that regulates genes involved with high affinity copper transport. Our results expand our understanding of how NMD regulates mRNAs from the same functional group and how the environment influences this regulation.
Assuntos
Cobre/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Códon sem Sentido , FMN Redutase/genética , Regulação Fúngica da Expressão Gênica , Homeostase , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3'-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3'-UTRs and are sensitive to NMD, although the extent by which these long 3'-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3'-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3'-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3'-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Organismos Geneticamente Modificados , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas SLC31 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genéticaRESUMO
The nonsense-mediated mRNA decay (NMD) pathway is a specialized mRNA degradation pathway that degrades select mRNAs. This pathway is conserved in all eukaryotes examined so far, and it triggers the degradation of mRNAs that prematurely terminate translation. Originally identified as a pathway that degrades mRNAs with premature termination codons as a result of errors during transcription, splicing, or damage to the mRNA, NMD is now also recognized as a pathway that degrades some natural mRNAs. The degradation of natural mRNAs by NMD has been identified in multiple eukaryotes, including Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana, and humans. S. cerevisiae is used extensively as a model to study natural mRNA regulation by NMD. Inactivation of the NMD pathway in S. cerevisiae affects approximately 10% of the transcriptome. Similar percentages of natural mRNAs in the D. melanogaster and human transcriptomes are also sensitive to the pathway, indicating that NMD is important for the regulation of gene expression in multiple organisms. NMD can either directly or indirectly regulate the decay rate of natural mRNAs. Direct NMD targets possess NMD-inducing features. This minireview focuses on the regulation of natural mRNAs by the NMD pathway, as well as the features demonstrated to target these mRNAs for decay by the pathway in S. cerevisiae. We also compare NMD-targeting features identified in S. cerevisiae with known NMD-targeting features in other eukaryotic organisms.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Animais , Arabidopsis/genética , Drosophila melanogaster/genética , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
The eukaryotic nonsense-mediated mRNA decay pathway (NMD) is a specialized pathway that contributes to the recognition and rapid degradation of mRNA with premature termination codons. In addition to mRNAs containing premature termination codons, NMD degrades non-nonsense-containing, natural mRNAs. Approximately 5-10% of the total Saccharomyces cerevisiae transcriptome is affected when NMD is inactivated. The regulation of natural mRNAs by NMD has physiological consequences. However, the physiological outcomes associated with the degradation of specific natural mRNAs by NMD are not fully understood. Here, we examined the physiological consequences resulting from the NMD-mediated regulation of an mRNA involved in copper homeostasis, in an attempt to understand why nmd mutant strains are more tolerant of toxic copper levels than wild-type yeast strains. We found that wild-type (UPF1) and upf1Δ mutants accumulate similar amounts of total copper when grown in medium containing elevated levels of copper; however, the copper levels in the cytoplasm of wild-type yeast cells were higher than in the upf1Δ mutant. Copper tolerance by the upf1Δ mutant is dependent on the presence of CTR2. Deletion of CTR2 resulted in similar cytoplasmic copper levels in wild-type and upf1Δ mutant strains, regardless of the environmental copper levels. This suggests that CTR2 plays a role in regulating the level of copper in the cytoplasm. We also found that the upf1Δ mutant contained elevated copper levels in the vacuole relative to wild-type yeast cells, after both strains were exposed to elevated copper levels.
Assuntos
Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Regulação Fúngica da Expressão Gênica , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Códon sem Sentido , Cobre/análise , Cobre/toxicidade , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter , Homeostase , RNA Helicases/metabolismo , RNA Mensageiro/genética , Proteínas SLC31 , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Vacúolos/metabolismoRESUMO
The eukaryotic nonsense-mediated mRNA (NMD) is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons, and importantly some natural mRNAs as well. Natural mRNAs with atypically long 3'-untranslated regions (UTRs) are degraded by NMD in Saccharomyces cerevisiae. A number of S. cerevisiae mRNAs undergo alternative 3'-end processing producing mRNA isoforms that differ in their 3'-UTR lengths. Some of these alternatively 3'-end processed mRNA isoforms have atypically long 3'-UTRs and would be likely targets for NMD-mediated degradation. Here, we investigated the role NMD plays in the regulation of expression of CTR2, which encodes a vacuolar membrane copper transporter. CTR2 pre-mRNA undergoes alternative 3'-end processing to produce two mRNA isoforms with 300-nt and 2-kb 3'-UTRs. We show that both CTR2 mRNA isoforms are differentially regulated by NMD. The regulation of CTR2 mRNA by NMD has physiological consequences, since nmd mutants are more tolerant to toxic levels of copper relative to wild-type yeast cells and the copper tolerance of nmd mutants is dependent on the presence of CTR2.
Assuntos
Cobre/farmacologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Poluentes Ambientais/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mutação , Isoformas de Proteínas/genética , Proteínas SLC31 , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The nonsense-mediated mRNA decay (NMD) pathway, present in most eukaryotic cells, is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and, importantly, some wild-type mRNAs. Earlier studies demonstrated that aberrant mRNAs with artificially extended 3'-untranslated regions (3'-UTRs) are degraded by NMD. However, the extent to which wild-type mRNAs with long 3'-UTRs are degraded by NMD is not known. We used a global approach to identify wild-type mRNAs in Saccharomyces cerevisiae that have longer than expected 3'-UTRs, and of these mRNAs tested, 91% were degraded by NMD. We demonstrate for the first time that replacement of the natural, long 3'-UTR from wild-type PGA1 mRNA, which encodes a protein that is important for cell wall biosynthesis, with a short 3'-UTR renders it immune to NMD. The natural PGA1 3'-UTR is sufficient to target a NMD insensitive mRNA for decay by the NMD pathway. Finally, we show that nmd mutants are sensitive to Calcofluor White, which suggests that the regulation of PGA1 and other cell wall biosynthesis proteins by NMD is physiologically significant.
Assuntos
Regiões 3' não Traduzidas/química , Códon sem Sentido , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Actinas/genética , Regulação Fúngica da Expressão Gênica , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Farneseno Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Mutação , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The nonsense-mediated mRNA decay (NMD) pathway regulates mRNAs that aberrantly terminate translation. This includes aberrant mRNAs and functional natural mRNAs. Natural mRNA degradation by NMD is triggered by mRNA features and environmental cues. Saccharomyces cerevisiae encodes multiple proteins with ferric and cupric reductase activity. Here, we examined the regulation by NMD of two mRNAs, FRE1 and FRE2, encoding ferric and cupric reductases in S. cerevisiae. We found that FRE2 mRNAs are regulated by NMD under noninducing conditions and that the FRE2 3'-UTR contributes to the degradation of the mRNAs by NMD. Conversely, FRE1 mRNAs are not regulated by NMD under comparable conditions. These findings suggest that regulation of functionally related mRNAs by NMD can be differential and conditional.
Assuntos
FMN Redutase/química , FMN Redutase/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Regiões 3' não Traduzidas , Cobre/metabolismo , Ferro/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
Colonization by the fungal pathogen Candida albicans varies significantly, depending upon the pH and availability of oxygen. Because of our interest in extracellular molecules as potential quorum-sensing molecules, we examined the physiological conditions which regulate the production of the aromatic alcohols, i.e., phenethyl alcohol, tyrosol, and tryptophol. The production of these fusel oils has been well studied for Saccharomyces cerevisiae. Our data show that aromatic alcohol yields for C. albicans are determined by growth conditions. These conditions include the availability of aromatic amino acids, the pH, oxygen levels, and the presence of ammonium salts. For example, for wild-type C. albicans, tyrosol production varied 16-fold merely with the inclusion of tyrosine or ammonium salts in the growth medium. Aromatic alcohol production also depends on the transcription regulator Aro80p. Our results are consistent with aromatic alcohol production-aromatic transaminases (gene products for ARO8 and ARO9), aromatic decarboxylase (ARO10), and alcohol dehydrogenase (ADH)-via the fusel oil pathway. The expression of ARO8, ARO9, and ARO10 is also pH dependent. ARO8 and ARO9 were alkaline upregulated, while ARO10 was alkaline downregulated. The alkaline-dependent change in expression of ARO8 was Rim101 independent, while the expression of ARO9 was Rim101 dependent.
Assuntos
Álcoois/metabolismo , Candida albicans/metabolismo , Álcool Desidrogenase/metabolismo , Aminoácidos Aromáticos/metabolismo , Carboxiliases/metabolismo , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Redes e Vias Metabólicas , Oxigênio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Transaminases/metabolismoRESUMO
mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell's specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.
Assuntos
Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fenantrolinas/farmacologia , RNA Polimerase II/metabolismo , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The nonsense-mediated mRNA decay (NMD) pathway is a specialized pathway that triggers the rapid degradation of select mRNAs. Initially, identified as a pathway that degrades mRNAs with premature termination codons, NMD is now recognized as a pathway that also regulates some natural mRNAs. Since natural mRNAs do not typically contain premature termination codons, these mRNAs contain features that target them to NMD. In Saccharomyces cerevisiae mRNAs with atypically long 3'-UTRs are usually degraded by NMD, however in some conditions a constitutively expressed SSY5 mRNA with multiple NMD targeting signals including an atypically long 3'-UTR is an exception. We investigated the features of the SSY5 mRNAs that confer immunity to NMD. We found that the SSY5 mRNA 3'-UTRs are sufficient to target NMD insensitive mRNA to the pathway. Replacing the SSY5 3'-UTRs with the cyc1-512 3'-UTRs, known to target mRNAs to NMD or with the CYC1 3'-UTR, known not to target mRNAs to NMD, resulted in production of SSY5 mRNAs that were regulated by NMD. These observations suggest that the SSY5 mRNAs require sequences both within the 5'-UTR and/or ORF as well as the 3'-UTR to escape decay by NMD.
RESUMO
Nonsense-mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild-type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady-state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5' and 3' untranslated region (UTR) lengths, and poly(A) tail length.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Saccharomyces cerevisiae/genética , Técnicas Genéticas , RNA Fúngico/químicaRESUMO
A method for determining mRNA half-lives in the polymorphic fungus Candida albicans is described. It employs growth in a defined medium, the inhibition of transcription with thiolutin (10-20 microg/mL), and quantitative Northern blotting. The method is effective for the A72, SC5314, and CAI-4 strains of C. albicans, and for mRNAs that have a wide variety of decay rates and steady-state abundances. The range of half-lives detected (from 4-168 min) shows that this method is effective for mRNAs with widely varying half-lives. The mRNA decay rates obtained are compared with those for orthologous mRNAs from Saccharomyces cerevisiae. This procedure should work for a broad range of C. albicans strains and can be adapted to other fungal species.
Assuntos
Candida albicans/genética , Candida albicans/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Primers do DNA/genética , Meia-Vida , Pirrolidinonas/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The dimorphic fungus Candida albicans produces farnesol as a quorum-sensing molecule that regulates cellular morphology. The biosynthetic origin of farnesol has been resolved by treating these cells with zaragozic acid B, a potent inhibitor of squalene synthase in the sterol biosynthetic pathway. Treatment with zaragozic acid B leads to an eightfold increase in the amount of farnesol produced by C. albicans. Furthermore, C. albicans cell extracts contain enzymatic activity to convert [(3)H]farnesyl pyrophosphate to [(3)H]farnesol. Many common antifungal antibiotics (e.g., zaragozic acids, azoles, and allylamines) target steps in sterol biosynthesis. We suggest that the fungicidal activity of zaragozic acid derives in large part from the accumulation of farnesol that accompanies the inhibition of sterol biosynthesis.