Here 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 5′ 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).
While Eap1p has a role in the rapid decay of Vts1p target transcripts it does not appear to play a role in regulating their translation. For example, we have been unable to document a role for Vts1p in regulating translation that is independent of transcript decay. For example, we have shown that GFP protein levels from the GFP-SRE+
mRNA are repressed approximately four fold compared to GFP-SRE-
mRNA and that this repression is eliminated in a vts1Δ
strain ( 
, and data not shown). However, when GFP protein levels were corrected using the levels of GFP mRNA we did not see a statistically significant difference in the ratio of GFP protein levels from the GFP-SRE+
mRNAs in wild-type, vts1Δ
cells (data not shown). These results suggest that repression of GFP-SRE+
reporter expression is mediated largely through degradation of target mRNAs, however we cannot rule out some role for translational repression mediated by Vts1p and/or Eap1p that our assays are not sensitive enough to detect. Alternatively, since the poly(A) tail is known to stimulate translation, Vts1p-mediated deadenylation of target mRNAs could simultaneously repress translation and induce transcript decay; if so, the two processes would not be separable.
A lack of a role for Eap1p in translational repression of Vts1p target mRNAs is consistent with the fact that a genome-wide survey of mRNAs that are translationally regulated by Eap1p show no statistically significant overlap with mRNAs that are bound by Vts1p 
. Thus the pool of Eap1p target mRNAs that are translationally regulated are distinct from the pool of Vts1p target mRNAs that are degraded. Since a poly(A) tail is required for mRNA translation, perhaps Vts1p-mediated deadenylation represses mRNA translation to the point where Eap1p can no longer augment this effect. In contrast, Eap1p is able to enhance decapping mediated by Vts1p-mediated deadenylation.
The Role of Eap1p in mRNA Decapping
What is the molecular mechanism that underlies the role of Eap1p in decapping? The mRNA cap structure stimulates translation through the ability of eIF4E to recruit the translation machinery to an mRNA through the eIF4E/eIF4G interaction. A critical step in mRNA decapping is thought to involve removal of the translation initiation complex from the mRNA. This model is based on results arguing that the decapping and translation machineries compete with one another for access to mRNAs 
. For example, disruption of eIF4G function in yeast results in increased rates of decapping 
. We find that the eIF4E-binding motif in Eap1p is required for its role in Vts1p-mediated degradation. Thus the eIF4E/Eap1p interaction may enhance decapping by destabilizing the translation initiation complex through displacement of eIF4G from the mRNA.
Eap1p may also enhance decapping by facilitating the recruitment of decapping factors to the 5′ end of the mRNA. This is supported by systematic efforts that identified protein complexes in S. cerevisiae
, including an interaction between Eap1p and the decapping activator Dhh1p 
. In this model the Eap1p/eIF4E interaction would serve to recruit Dhh1p to the vicinity of the cap thereby facilitating Dhh1p-mediated decapping. However this model may not be applicable to Vts1p target mRNAs as Vts1p-mediated decay of the GFP-SRE+
mRNA does not involve Dhh1p 
The Function of Eap1p in Yeast
Eap1p functions in a variety of biological processes including pseudohyphal growth 
, the response to oxidative stress induced by exposure to diamide and cadmium 
, the response to vesicular transport defects 
and the regulation of rapamycin-induced inactivation of TOR-signaling 
. In addition, polysome analysis has shown that Eap1p regulates the translation of specific target transcripts 
. Taken together with our data, these results suggest that Eap1p regulates the translation and/or stability of specific mRNAs under a variety of physiological conditions and that its interaction with RNA-binding proteins such as Vts1p (this study), Puf1 and Puf2 
as well as Puf5 (Aaron Goldstrohm, University of Michigan, personal communication), at least in part, controls which transcripts Eap1p regulates.
The Role of eIF4E-binding Proteins in mRNA Decay
Another eIF4E-binding protein, known as 4E-transporter (4E-T), has been shown to play a role in transcript decay in mammalian cells 
. Originally identified as being responsible for transporting eIF4E into the nucleus 
, subsequent experiments showed that depletion of 4E-T from HeLa cells resulted in significant stabilization of A/U rich element (ARE)-containing mRNAs 
. It is thought that 4E-T may regulate the transition from translational repression to mRNA degradation through a mechanism involving the localization of mRNAs to P-bodies, which are sites of mRNA storage and decay 
. While the role of the 4E-T/eIF4E interaction in ARE-mediated decay has not been assessed, indirect evidence suggests that 4E-T may also stimulate decapping. Though both are eIF4E-binding proteins, Eap1p and 4E-T are not homologs. Thus, these data suggest that other eIF4E-binding proteins might play a role in stimulating transcript decapping through their interaction with eIF4E.