Cell Morphogenesis Proteins Are Translationally Controlled through UTRs by the Ndr/LATS Target Ssd1
Figure 6
The 3′UTR of SIM1 is required for efficient Ssd1-SIM1 mRNA interaction but not translational control.
(A) Diagram of wild-type SIM1 locus (top) and SIM1 locus with its endogenous 3′UTR replaced with the CYC1 3′UTR (bottom). (B) SIM1 mRNA immunoprecipitation of wild-type (SIM1-SIM13′UTR) or 3′UTR-ablated (SIM1-CYC13′UTR) locus, normalized to the pull-down of the positive control SUN4. Three replicate experiments showed a significant difference in the ability of Ssd1 to immunoprecipitate SIM1 RNA when its endogenous 3′UTR is present compared to when its 3′UTR has been replaced. Ssd1-mRNA immunoprecipitation efficiency was evaluated by comparing SIM1 enrichment in antibody-treated over antibody-untreated samples and corrected for experimental variability by normalization to the positive control SUN4. Error bars represent ± SEM and * indicates P-value 0.01 to 0.05 at 95% confidence intervals as calculated by unpaired two-tailed Student's t-test. (C) Ablating the endogenous 3′UTR of the SIM1 does not disrupt phosphoregulated Ssd1 translational control over Sim1. Western blotting against secreted and internal Sim1 was performed as described in Materials in Methods, from cells harvested at 5, 15 and 30 minutes following media replenishment. In both wild-type (left panels) and 3′UTR-ablated SIM1 (right panels), secreted Sim1 levels are depleted when Ssd1 is hyperactivated (see cbk1-as SSD1 +1NA-PP1). Translational repression of Sim1 expressed from both loci is dependent on the presence of Ssd1 (see cbk1-as ssd1Δ +1NA-PP1) 1NA-PP1 treatment (see +DMSO lanes).