Long 3’-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

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

Long 3’-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

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

Copper Tolerance of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e Nonsense-Mediated mRNA Decay Mutants

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

Immunity of the \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e SSY5 mRNA to nonsense-mediated mRNA decay

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 a typically 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 conferimmunity 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

Long 3′-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in Saccharomyces cerevisiae

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

Long 3’-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

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

Determination of mRNA half-lives in Candida albicans using thiolutin as a transcription inhibitor

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 μg/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. Une méthode pour déterminer les demi-vies d\u27ARNm dans le champignon polymorphe Candida albicans est décrite. Il utilise la croissance dans un milieu défini, l\u27inhibition de la transcription avec la thiolutine (10–20 μg / mL) et le Northern blot quantitatif. La méthode est efficace pour les souches A72, SC5314 et CAI-4 de C. albicans, et pour les ARNm qui ont une grande variété de taux de désintégration et d\u27abondances à l\u27état d\u27équilibre. La gamme de demi-vies détectées (de 4 à 168 min) montre que cette méthode est efficace pour les ARNm avec des demi-vies très variables. Les taux de désintégration des ARNm obtenus sont comparés à ceux des ARNm orthologues de Saccharomyces cerevisiae. Cette procédure devrait fonctionner pour une large gamme de souches de C. albicans et peut être adaptée à d\u27autres espèces fongiques

Farnesol Biosynthesis in \u3ci\u3eCandida albicans\u3c/i\u3e: Cellular Response to Sterol Inhibition by Zaragozic Acid B

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 [3H]farnesyl pyrophosphate to [3H]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

Copper Tolerance of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e Nonsense-Mediated mRNA Decay Mutants

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