The decay of wild-type PPR1 mRNA is Upf1p-dependent. (A) PPR1 mRNA accumulation in BY4741 (ppr1Δ) grown in YPD, and PLY167 [pAA79] (UPF1) and PLY167 [pRS315] (upf1Δ) grown in CM-leucine. Northern blots were prepared with 15 µg of total RNA and hybridized with radiolabeled PPR1, CYH2 and ScR1 DNA. CYH2 pre-mRNA is a target for NMD used to confirm the NMD phenotype of our yeast cells (18). ScR1 mRNA is transcribed by RNA polymerase III and is not degraded by NMD (33,34). It is used as a loading control. The relative PPR1 steady-state levels are indicated under the northern blot hybridized with radiolabeled PPR1 DNA. (B) PPR1 mRNA half-lives were measured in isogenic yeast strains AAY334 (UPF1, upper image) and AAY335 (upf1Δ, lower image) both grown in YAPD. The PhosphorImages are of representative northern blots hybridized with radiolabeled PPR1 DNA. The northern blots were prepared with 15 µg total RNA harvested at different time points following inhibition of RNA polymerase II. The 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 point zero. The percent mRNA remaining versus time after transcriptional inhibition was plotted using Sigmaplot™. The PPR1 mRNA half-lives was determined and is shown to the right of the PhosphorImages.

Wild-type PPR1 mRNA also accumulates in upf2Δ, upf3Δ, dcp1Δ and xrn1Δ cells. (A) PPR1 mRNA accumulation in HFY1200 (wild-type), HFY870 (upf1Δ), HFY1300 (upf2Δ) and HFY861 (upf3Δ) yeast cells grown in YAPD. (B) PPR1 mRNA accumulation in yeast cells HFY1200 (wild-type), HFY1067 (dcp1Δ) and HFY1081 (xrn1Δ) grown in YAPD. The northern blots were prepared using 15 µg of total RNA and hybridized with radiolabeled PPR1, CYH2 and ScR1 DNA. The relative PPR1 steady-state levels are indicated under the northern blots hybridized with radiolabeled PPR1 DNA.

Wild-type PPR1 mRNA accumulates in hrp1-3 yeast cells at the non-permissive temperature. PPR1 mRNA accumulation in Y137 (wild-type) and Y191 (hrp1-3) at room temperature (rt) and after 1 h at 37°C. Y137 and Y191 were grown in YAPD. Northern blots were prepared using 15 µg of total RNA and hybridized with radiolabeled PPR1, PGK1 and ScR1 DNA. The relative PPR1 steady-state levels are indicated under the northern blot hybridized with radiolabeled PPR1 DNA.

Schematic diagrams of the mRNAs used to map the PPR1 mRNA UDE, and representative northern blots of the mRNA encoded by each construct in W303a (UPF1) and AAY320 (upf1Δ) yeast strains. The location of the 5′-UTR, 3′-UTR and ORF of PPR1 and ACT1 mRNAs are indicated. The first base of the ORF is +1. The fusion junction locations are indicated in nucleotides relative to the first base of the ORF. Construct 1, PPR1-ACT1, is an in-frame fusion of the PPR1 5′-UTR and the first 1255 nt of the PPR1 ORF fused to nucleotides +581 to +1128 of the ACT1 ORF and ACT1 3′-UTR. Construct 2, ACT1-PPR1, includes the 5′-UTR and nucleotides +1 to +595 of ACT1 fused in frame to nucleotides +1250 to +2715 of the PPR1 ORF and the PPR1 3′-UTR. Construct 3, mini-PPR1 gene, was constructed by deleting +93 to +2156 of the PPR1 ORF. Construct 4, ACT1 5′-UTR mini-PPR1 gene, is identical to construct 3 except the 5′-UTR of PPR1 has been replaced with the 5′-UTR of ACT1. Construct 5, PPR1 5′-UTR ACT1-PPR1, is identical to construct 2 except the ACT1 5′-UTR has been replaced with the 5′-UTR of PPR1. The abundance of the construct mRNAs in isogenic UPF1 and upf1Δ yeast strains grown in CM- leucine was determined by northern blotting. The relative construct mRNA steady-state levels are indicated under the northern blots. The northern blots were prepared using 15 µg of total RNA and hybridized with radiolabeled PPR1, ACT1, CYH2 (data not shown) and ScR1 (data not shown) DNA.

mRNAs encoded by the PPR1-ACT1 and the mini-PPR1 gene constructs are degraded more rapidly in AAY334, a UPF1 yeast strain, than the isogenic upf1Δ yeast strain, AAY335. mRNA half-lives for PPR1-ACT1 (A) and PPR1 mini-mRNA (B) were determined from northern blots of total RNA harvested from UPF1 and upf1Δ yeast cells grown in CM-leucine at time points following the arrest of transcription as described in the legend of Figure 1B. The time points at which the cells were harvested after inhibition of transcription are shown above the PhosphorImages and are different for the mRNAs encoded by the PPR1-ACT1 construct and the mini-PPR1 gene construct. The upper band in the PhosphorImages of (A) is the endogenous PPR1 mRNA.

Summary of construct analysis. The UDE maps to a region within the 5′ end to nucleotide +92 of the PPR1 mRNA. Nucleotides +93 to +2715 of the PPR1 ORF and the PPR1 3′-UTR do not contain an UDE. The schematic diagrams represent PPR1 mRNA (top) and the relevant portions of PPR1 mRNA in the five constructs used to map the location of the UDE. The diagrams are numbered relative to the PPR1 transcription start point at +1.