Nonsense-mediated mRNA decay of the ferric and cupric reductase mRNAs FRE1 and FRE2 in Saccharomyces cerevisiae

Abstract

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.

Keywords: FRE1-2 mRNA; Saccharomyces cerevisiae; biometal homeostasis; mRNA decay; nonsense-mediated mRNA decay.

Figure 1.. FRE1 mRNA is immune to NMD mediated degradation whereas, FRE2 mRNAs isoforms are differentially regulated by NMD under inducing and non-inducing conditions

Schematic representations of FRE1 (A) and the FRE2 mRNA isoforms (B) showing the Upstream open reading frame (uORF), the open triangle indicates the potential −1 programmed ribosomal frameshift (−1PRF) and the atypically long 3′-UTRs. Representative mRNA steady-state accumulation levels (C, D, E and F). Steady-state accumulation levels were measured with total RNA from wild-type strain W303 (UPF1) [25], and NMD mutants (upf1Δ) [18], HFY1300 (upf2Δ) [26], HFY861 (upf3Δ)[27] [18]. The northern blots were probed with DNA specific to the FRE1, FRE2 and COX19 (1F) open reading frames (ORFs). The major FRE2 mRNA isoform fold change (upf1Δ/UPF1) are shown to the right of the northern blots (C, D, and F). FRE1 mRNA fold change (upf1Δ/UPF1) are shown to the right of northern blots (E and F) and COX19 mRNA fold change (upf1Δ/UPF1) is shown to the right of northern blot (F). The fold change shown to the right of 1D and 1E is for low copper conditions, while the fold changes to the right of 1F are for low iron conditions. CYH2 and SCR1 were used as controls. CYH2 pre-mRNA was used as an NMD control because CYH2 pre-mRNA is degraded by NMD. SCR1 was used as a loading control for all northern blots. SCR1 is an RNA polymerase III transcript that is not regulated by NMD.

Figure 2.. Decay of FRE2 mRNAs in wild-type and NMD mutant strains is responsive to copper availability.

Representative half-life northerns of FRE2 mRNA. The mRNA half-lives were measured with total RNA extracted from wild-type strain AAY334 (UPF1 rpb1–1)[18] and NMD mutant strain AAY335 (upf1Δ rpb1–1)[18] grown under 100 μM Bathocuproinedisulfonic acid (BCS), low copper (A, B) and in 100 μM copper (C, D). Yeast cells were harvested at eight time points over 35 minutes. Individual time points are indicated above the half-life northerns. The half-lives were determined using SigmaPlot and are shown to the right of the northerns. All half-life measurements are an average of at least two independent experiments. CYH2 and SCR1 were used as controls as described in Figure 1.

Figure 3.. Decay of FRE1 mRNA in wild-type and NMD mutant strains is responsive to copper availability.

Representative half-life northerns of FRE1 mRNA. The mRNA half-lives were measured with total RNA extracted from wild-type strain AAY334 (UPF1 rpb1–1)[18] and NMD mutant strain AAY335 (upf1Δ rpb1–1)[18] grown under low copper (A, B) and in 100 μM copper (C, D). Yeast cells were harvested at eight time points over 35 minutes. Individual time points are indicated above the half-life northerns. The half-lives were determined using SigmaPlot and are shown to the right of the northerns. All half-life measurements are an average of at least two independent experiments. CYH2 and SCR1 were used as controls as described in Figure 1.

Figure 4.. The FRE2 3’-UTR is not sufficient to target an NMD insensitive mRNA to the pathway but it contributes to the degradation of the mRNAs by the pathway

Schematic representation of CYC1FRE2 3’-UTR (A) and FRE2CYC1 3’-UTR fusion mRNAs (B). Representative mRNA half-life northern blots of CYC1FRE2 3’-UTR (C and E, left panels) in wild-type (UPF1 rpb1–1), and NMD mutants (upf1Δ rpb1–1) respectively. The northerns were probed with DNA specific to the FRE2 3’-UTR. Representative mRNA half-life northern blots of FRE2CYC1 3’-UTR mRNA (D and F right panels) in wild-type (UPF1 rpb1–1), and NMD mutants (upf1Δ rpb1–1) respectively. The northern blots were probed with DNA specific to the FRE2 5’-UTR and ORF. All yeast cells were grown in complete minimal media lacking leucine, and were harvested at eight time points over thirty-five minutes after transcription inhibition. Specific time points are indicated above the half-life northern blots and the half-lives are shown to the right of the northern blots. The half-lives were determined using SigmaPlot. CYH2 and SCR1 are used as controls as described in Figure 1.