Figure 2.

Ebs1 participates in NMD in a way similar to human Smg proteins. (A and B) Total RNA from log phase yeast grown in YPD was prepared from wt, upf1Δ, ebs1Δ, est1Δ, ebs1Δest1Δ and ebs1Δupf1Δ cells and subjected to northern blot analysis using a probe which detects both the CYH2 pre-mRNA and CYH2 mRNA. In (A) the filter was stripped and re-probed to detect the ade2-1 mRNA. CYH2 pre-mRNA levels (CYH2 ratio) and ade2-1 mRNA levels (ade2-1 ratio) are expressed relative to wild-type cells, after normalization using the CYH2 mRNA signal. (C) HeLaβG^WT and HeLaβG^NS39 cells (see Materials and Methods section) were transfected with shRNA vectors against human Smg7 (sh7^A and sh7^B), human Smg6 (sh6) and human Upf1 and with empty vector controls (shEV). Five days after transfection, total mRNA was prepared and the levels of β-globin mRNA were determined on a northern blot. Alternatively, the same cell lines were transfected with vectors expressing a Flag-tagged version of human Smg7 (F-Smg7) or empty vector controls (F-EV) and processed 48 h after transfection. The signal of βG^NS39 (βGL ratio) is expressed relative to empty vector-transfected cells, after normalization using the actin signal (loading control) and the signal of βG^WT. (D) Wild-type yeasts were transformed with centromeric plasmids harboring EBS1 and Smg7 under the constitutive ADH promoter, or with empty vector alone. Total RNA was probed using the CYH2 probe as in A–C.