Figure 2.

Gbp2 and Hrb1 are involved in translation repression of NMD targets. (A) Translation of the DBP2^PTC reporter results in a truncated protein, shown on a western blot. Zwf1 served as a loading control. (B) Translation of the 5ʹ-proximal PTC-containing CBP80^PTC reporter results in the expression of the full-length protein, shown on a western blot. (C-F) Proper translational repression of the DBP2^PTC requires both Gbp2 and Hrb1 and proper translational repression of the CBP80^PTC requires Gbp2. Expression of DBP2^PTC (C) and CBP80^PTC (E) in the indicated strains was monitored by western blot analysis. (D, F) Protein expression of independent experiments shown in (C) and (E) were quantified. MYC-Dbp2^PTC (D) and MYC-Cbp80^PTC (F) signals were normalized to the loading control and the relative reporter RNA level (Fig S2B, S2C). The standard deviation of upf1Δ cells is 4.1 and 6.5, respectively. n = 5. (G) The translational repression activity of Gbp2 and Hrb1 requires Upf1. Expression of the PTC-containing reporter transcripts is shown in upf1∆ and upf1∆ gbp2∆ hrb1∆ cells. The asterisk indicates a band of Gbp2. (H) Protein expression shown in (G) was quantified as in (D) and (F). n = 4. (I) Known RGG motif translational repressors do not suppress translation of PTC-containing transcripts. Expression of the CBP80^PTC was compared in the indicated strains on western blots. See also Fig S2E. (J) Protein level of three independent experiments, one of which is shown in (I), was quantified. MYC-Cbp80^PTC signals were normalized to the loading control Zwf1. Results for gbp2Δ and upf1Δ are replotted from previous experiments for comparison (Fig S2C)