S indicated in either vts1D cells (A) or eap1D cells (B) and crude extracts were subjected to immunoprecipitation using an anti-Flag resin. Starting crude extracts (input) and the resulting immunoprecipitates (IP:aFLAG) were analyzed by Western blot. Input samples represent 5 for Vts1p blots, 2 for Eap1p blots and 1 for eIF4E of material used in the immunoprecipitations. The use of the Vts1p RNA binding mutant (Vts1pRBD2) and Eap1p mutant defective for eIF4E binding (Eap1pmt) are as indicated. doi:10.1371/journal.pone.0047121.g(Eap1pmt-HA) [29]. When HIF-2��-IN-1 cost Eap1p-HA was expressed in the eap1D strain, GFP-SRE+ mRNA decayed at a rate similar to wild-type (Figure 7A and C). However when Eap1pmt-HA was expressed in the eap1D strain, the decay rate of GFP-SRE+ mRNA was not rescued to the wild-type rate. Western blot analysis confirmed that both Eap1p-HA and Eap1pmt-HA were expressed at similar levels and therefore differences in protein expression cannot account for the observed difference in mRNA decay kinetics (Figure 7B). These data demonstrate that a functional Eap1p eIF4E-binding motif is required for the rapid degradation of GFP-SRE+ mRNA.DiscussionHere we demonstrate a role for Eap1p in 
Vts1p-mediated mRNA decay. The ability of Eap1p to interact with the cap binding protein eIF4E is required for this function and, consistent with its proximity to the 59 cap, Eap1p stimulates mRNA decapping. While Eap1p is required for efficient decay of a reporter mRNA bearing wild-type SREs, it is not required for degradation of a reporter bearing mutated non-functional SREs. This suggests a specific role for Eap1p in the degradation of Vts1p target transcripts. In addition, we show that Vts1p interacts with Eap1p and that Eap1p is able to mediate an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby recruitment of Eap1p to target transcripts, through its interaction with Vts1p, stimulates decapping via binding to eIF4E. Interestingly, another RNA binding protein, Puf5p, also 57773-63-4 site Functions with Eap1p to enchance the decapping of its target mRNAs (Aaron Goldstrohm, University of Michigan, personal communication).Binding to eIF4E is Required for Eap1p to Stimulate Transcript DecayTo explore the molecular mechanism that underlies the role of Eap1p in the decay of Vts1p target transcripts we asked whether the Eap1p/eIF4E interaction was required for mRNA degradation. This involved assaying the stability of GFP-SRE+ mRNA by transcriptional pulse-chase in eap1D cells expressing either wildtype Eap1p (Eap1p-HA) or a version of Eap1p harboring the Y109A;L114A mutations that disrupt its eIF4E-binding abilityEap1p Functions in Vts1p-Mediated Transcript DecayFigure 7. The Eap1p/eIF4E interaction is required for efficient decay of Vts1p target mRNAs. eap1D cells were rescued with either wildtype Eap1p-HA or a mutant version of Eap1p that is defective in its ability to interact with eIF4E (Eap1pmt-HA) and 12926553 the stability of GFP-SRE+ mRNA was assessed in these cells using a transcriptional pulse-chase approach and Northern blot (A) as described in Figure 1. Western blot (B) was used to compare the expression of wild-type Eap1p-HA to that of Eap1pmt-HA using the PSTAIR protein as a loading control. The results of at least 3 independent experiments were quantitated and normalized using the levels of SCR1 RNA and graphed with error bars representing standard deviation (C). Data from wild-type and eap1D cells is 
from Figure 1A. doi:1.S indicated in either vts1D cells (A) or eap1D cells (B) and crude extracts were subjected to immunoprecipitation using an anti-Flag resin. Starting crude extracts (input) and the resulting immunoprecipitates (IP:aFLAG) were analyzed by Western blot. Input samples represent 5 for Vts1p blots, 2 for Eap1p blots and 1 for eIF4E of material used in the immunoprecipitations. The use of the Vts1p RNA binding mutant (Vts1pRBD2) and Eap1p mutant defective for eIF4E binding (Eap1pmt) are as indicated. doi:10.1371/journal.pone.0047121.g(Eap1pmt-HA) [29]. When Eap1p-HA was expressed in the eap1D strain, GFP-SRE+ mRNA decayed at a rate similar to wild-type (Figure 7A and C). However when Eap1pmt-HA was expressed in the eap1D strain, the decay rate of GFP-SRE+ mRNA was not rescued to the wild-type rate. Western blot analysis confirmed that both Eap1p-HA and Eap1pmt-HA were expressed at similar levels and therefore differences in protein expression cannot account for the observed difference in mRNA decay kinetics (Figure 7B). These data demonstrate that a functional Eap1p eIF4E-binding motif is required for the rapid degradation of GFP-SRE+ mRNA.DiscussionHere we demonstrate a role for Eap1p in Vts1p-mediated mRNA decay. The ability of Eap1p to interact with the cap binding protein eIF4E is required for this function and, consistent with its proximity to the 59 cap, Eap1p stimulates mRNA decapping. While Eap1p is required for efficient decay of a reporter mRNA bearing wild-type SREs, it is not required for degradation of a reporter bearing mutated non-functional SREs. This suggests a specific role for Eap1p in the degradation of Vts1p target transcripts. In addition, we show that Vts1p interacts with Eap1p and that Eap1p is able to mediate an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby recruitment of Eap1p to target transcripts, through its interaction with Vts1p, stimulates decapping via binding to eIF4E. Interestingly, another RNA binding protein, Puf5p, also functions with Eap1p to enchance the decapping of its target mRNAs (Aaron Goldstrohm, University of Michigan, personal communication).Binding to eIF4E is Required for Eap1p to Stimulate Transcript DecayTo explore the molecular mechanism that underlies the role of Eap1p in the decay of Vts1p target transcripts we asked whether the Eap1p/eIF4E interaction was required for mRNA degradation. This involved assaying the stability of GFP-SRE+ mRNA by transcriptional pulse-chase in eap1D cells expressing either wildtype Eap1p (Eap1p-HA) or a version of Eap1p harboring the Y109A;L114A mutations that disrupt its eIF4E-binding abilityEap1p Functions in Vts1p-Mediated Transcript DecayFigure 7. The Eap1p/eIF4E interaction is required for efficient decay of Vts1p target mRNAs. eap1D cells were rescued with either wildtype Eap1p-HA or a mutant version of Eap1p that is defective in its ability to interact with eIF4E (Eap1pmt-HA) and 12926553 the stability of GFP-SRE+ mRNA was assessed in these cells using a transcriptional pulse-chase approach and Northern blot (A) as described in Figure 1. Western blot (B) was used to compare the expression of wild-type Eap1p-HA to that of Eap1pmt-HA using the PSTAIR protein as a loading control. The results of at least 3 independent experiments were quantitated and normalized using the levels of SCR1 RNA and graphed with error bars representing standard deviation (C). Data from wild-type and eap1D cells is from Figure 1A. doi:1.