Figure 1. MBS tagging may destabilize mRNAs through the NMD pathway in yeast.

(A) RT-qPCR of MBS-tagged mRNAs in wildtype (W) or Δupf1 (Δ) strain background. When measuring HAC1 mRNA abundance, cells were treated with 1 μg/ml tunicamycin for 2 hours. The mRNA levels were normalized to their corresponding wildtype (WT) mRNAs. P values (left to right) are 0.39, <0.0001, 0.93, 0.69, 0.0099, 0.60, 0.35, 0.0092, 0.12, 0.35, 0.13, 0.22. n = 3 biologically independent experiments. (B) Representative images of ATP2 mRNA (green) and mitochondria (magenta). ATP2 mRNAs were detected by smFISH. Mitochondria were labeled using mitochondria-targeted GFP. Images were max-Z projected. Scale bars are 2 μm. (C) Number of ATP2 mRNAs per cell as quantified from the smFISH images. n = 291 and 457 cells. P value is <0.0001. (D) RT-qPCR of ATP2 mRNAs tagged with 24x or 12xMBS array in the presence or absence of MCP-GFP. P values are <0.0001, 0.0002, <0.0001. n = 3 biologically independent experiments. (E) mRNA stability assay of the ATP2 mRNA in indicated strains. Cells were treated with phenanthroline to inhibit transcription. mRNA levels were measured by RT-qPCR. The fraction of remaining mRNAs was calculated by normalizing the ATP2 mRNA level to the level at time point 0 minute. A two-sided Student’s t-test was performed to compare WT and ATP2-24xMBS strains. P values are 0.012, 0.015, 0.0067. n = 3 biologically independent experiments. (F) Bar graph showing the normalized levels of Atp2 protein as quantified from Western blots. Pgk1 was used as a loading control. P value is 0.0002. n = 3 biologically independent experiments. (G, H) Serial dilutions of yeast cells were spotted on agar plates with indicated carbon source (G) or the ER protein folding stress inducer tunicamycin (H). In Figure 1, all error bars indicate mean ± SD. In Figure 1, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant (all analysis are two-sided Student’s t-test).