Gene set coregulated by the Saccharomyces cerevisiae nonsense-mediated mRNA decay pathway

Abstract

The nonsense-mediated mRNA decay (NMD) pathway has historically been thought of as an RNA surveillance system that degrades mRNAs with premature translation termination codons, but the NMD pathway of Saccharomyces cerevisiae has a second role regulating the decay of some wild-type mRNAs. In S. cerevisiae, a significant number of wild-type mRNAs are affected when NMD is inactivated. These mRNAs are either wild-type NMD substrates or mRNAs whose abundance increases as an indirect consequence of NMD. A current challenge is to sort the mRNAs that accumulate when NMD is inactivated into direct and indirect targets. We have developed a bioinformatics-based approach to address this challenge. Our approach involves using existing genomic and function databases to identify transcription factors whose mRNAs are elevated in NMD-deficient cells and the genes that they regulate. Using this strategy, we have investigated a coregulated set of genes. We have shown that NMD regulates accumulation of ADR1 and GAL4 mRNAs, which encode transcription activators, and that Adr1 is probably a transcription activator of ATS1. This regulation is physiologically significant because overexpression of ADR1 causes a respiratory defect that mimics the defect seen in strains with an inactive NMD pathway. This strategy is significant because it allows us to classify the genes regulated by NMD into functionally related sets, an important step toward understanding the role NMD plays in the normal functioning of yeast cells.

FIG. 1.

The increase in ATS1 mRNA accumulation seen in upf1Δ yeast strains relative to UPF1 yeast strains is an indirect effect of inactivation of the NMD pathway. (A) Representative Northern blot prepared with total RNAs from W303a (ATS1 UPF1), AAY320 (ATS1 upf1Δ), and AAY315 (ats1Δ UPF1). The Northern blots were hybridized with radiolabeled ATS1 (top), CYH2 (middle), and ScR1 (bottom) DNA probes. CYH2 and ScR1 are controls. CYH2 is a control for the NMD phenotype of the yeast strains. The CYH2 probe detects both CYH2 pre-mRNA and mRNA. CYH2 pre-mRNA is inefficiently spliced, and consequently a significant amount of this pre-mRNA is exported to the cytoplasm. CYH2 pre-mRNA has an in-frame stop codon within its intron that targets it for NMD, while CYH2 mRNA is not an NMD target (14). ScR1 is an RNA polymerase III transcript that is not degraded by NMD (29). It was used as a loading control. The relative ATS1 mRNA levels in UPF1 and upf1Δ yeast cells are shown below the corresponding bands in the top part of panel A. (B) Determination of ATS1 mRNA half-life by Northern blot analysis of total RNA harvested from isogenic yeast strains AAY334 (UPF1, upper image) and AAY335 (upf1Δ, lower image) at the indicated time points (in minutes) following arrest of transcription. Blots were hybridized with radiolabeled ATS1 DNA and PhosphorImaged. The half-lives (T_1/2, minutes) are the averages of at least three independent experiments and were determined from a plot of percent mRNA remaining versus time. Percent mRNA remaining was calculated by dividing the number of pixels contained in a particular band by the number of pixels contained in the first time point. The half-life of ATS1 mRNA in each yeast strain is to the right of the PhosphorImages.

FIG. 2.

Potential transcription factor binding sites in the ATS1 promoter region. (A) Schematic diagram of the putative transcription factor binding sites in the 500 bp upstream of the ATS1 ORF identified with the Promoter Database of Saccharomyces cerevisiae (http://rulai.cshl.edu/SCPD). The FUN30 ORF is on the opposite strand of the ATS1 ORF, and its position relative to putative transcriptional activator binding sites is indicated. (B) Comparison of the ATS1 promoter regions from S. cerevisiae, S. paradoxus, S. mikatae, and S. bayanus. The conserved potential binding sites in the ATS1 promoter region for Adr1, Gcr1, SCB, and Gal4 are shaded.

FIG. 3.

GAL4 and ADR1 mRNAs accumulate in upf1Δ cells relative to UPF1 cells. Representative Northern blots were prepared with total RNAs from W303a (UPF1), AAY320 (upf1Δ), PJ69-4a (gal4Δ), and Research Genetics strain 3573 (adr1Δ). The Northern blots were hybridized with radiolabeled GAL4 or ADR1 and CYH2 and ScR1 DNAs. The relative GAL4 and ADR1 mRNA levels in UPF1 and upf1Δ yeast cells are shown below the corresponding bands.

FIG. 4.

ATS1 expression is not increased under conditions that activate Gal4p. Shown is a representative Northern blot prepared with total RNAs from W303a (UPF1) and AAY320 (upf1Δ) grown in YP with 2% glucose (YP-2% glu) or 2% galactose (YP-2% gal) and from PJ69-4a (gal4Δ) grown in glucose. The Northern blots were hybridized with radiolabeled ATS1, GAL1, and ScR1 probes. The relative ATS1 mRNA levels are shown below the corresponding bands in the top panel.

FIG. 5.

Adr1 may be a transcription activator of ATS1. (A) Representative Northern blot prepared with total RNA from W303a transformed with pRS314 (ADR1) and pRS314ADR1 (CEN-ADR1). The Northern blots were hybridized with radiolabeled ATS1, ADR1, and ScR1 DNA probes. (B) Representative Northern blot prepared with total RNAs from W303a (UPF1), AAY320 (upf1Δ), 3575 (adr1Δ), and BY4741 (ADR1). The left column of images are the result of hybridization to Northern-blotted total RNA extracted from cells cultured under repressing conditions (YP-8% glucose [YP-8% glu]). The right column has images of hybridization to Northern blotted total RNA extracted from cells cultured under derepressing conditions (YP-3% ethanol-1% d-glucose [YP-ethanol]). The Northern blots were hybridized with radiolabeled ATS1, ADH2, and ScR1 DNA probes. Adr1 is a positive regulator of ADH2, so ADH2 mRNA levels are a control for repression and derepression of Adr1. The relative mRNA levels are shown below the corresponding bands.

FIG. 6.

Wild-type ADR1 mRNA also accumulates in upf2Δ, upf3Δ, dcp1Δ, and xrn1Δ cells. (A) Steady-state ADR1 mRNA levels in HFY1200 (UPF1 UPF2 UPF3; wild type), HFY870 (upf1Δ), HFY1300 (upf2Δ), and HFY861 (upf3Δ) yeast cells grown in YAPD. (B) Steady-state ADR1 mRNA levels in HFY1200 (wild type), HFY1067 (dcp1Δ), and HFY1081 (xrn1Δ) cells grown in YAPD. The Northern blots were prepared with total RNA and hybridized with ADR1, CYH2, and ScR1 DNA probes. The relative ADR1 mRNA levels are shown below the corresponding bands.

FIG. 7.

ADR1 mRNA accumulation following arrest of transcription. Northern blots were prepared with total RNAs harvested from isogenic yeast strains AAY334 (UPF1) and AAY335 (upf1Δ) at the indicated time points (minutes) after inhibition of RNA polymerase II. AAY334 and AAY335 carry rpb1-1, a temperature-sensitive allele coding for a component of RNA polymerase II. Transcription arrests rapidly in these strains when they are shifted to the nonpermissive temperature. (A, B) PhosphorImages of a representative Northern blot hybridized with radiolabeled ADR1 DNA (A) and then stripped and reprobed with radiolabeled CYH2 DNA (B). Essentially identical results were obtained in three independent experiments where transcription was arrested by a temperature shift and in one experiment in which thiolutin was used to arrest transcription. Inhibition of RNA polymerase II was effective in these experiments because CYH2 pre-mRNA levels decreased as expected following transcription arrest (B). (C) PhosphorImages of a representative Northern blot prepared with RNAs from W303a and AAY320 transformed with pKD34, which carries P_GAL10-ADR1-T_CYC1, and hybridized with radiolabeled ADR1 DNA. Percent mRNA remaining at each time point following inhibition of transcription was calculated by dividing the amount of probe hybridized to a particular band (corrected for loading with ScR1) by the amount of probe hybridized to the band at time zero. The percent mRNA remaining versus time after transcription inhibition was plotted with SigmaPlot.

FIG. 8.

Overexpression of ADR1 causes respiratory impairment. The yeast strains used were W303a (UPF1 ADR1) transformed with pRS314 (vector control), pRS314ADR1 (CEN-ADR1), and pMW5 (2μ-ADR1); AAY320 (upf1Δ) transformed with pRS314; BY4741 (ADR1); and Research Genetics strain 3575 (adr1Δ in BY4741). The strains were grown to an optical density at 260 nm of 0.4 to 0.6; diluted 10⁰, 10⁻¹, 10⁻², and 10⁻³; spotted onto complete minimal medium lacking tryptophan and containing either glucose (left panel) or lactate (right panel); and incubated at 18°C.