Figure 4.

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.