The eIF4E-Binding Protein Eap1p Functions in Vts1p-Mediated Transcript Decay

Sequence-specific RNA binding proteins can induce the degradation of mRNAs through their ability to recruit proteins that trigger transcript destabilization. For example, Vts1p, the S. cerevisiae member of the Smaug family of RNA binding proteins, is thought to induce transcript decay by recruiting the Ccr4p-Pop2p-Not deadenylase complex to target mRNAs. The resulting deadenylation triggers transcript decapping followed by 5′-to-3′ exonucleolytic decay. Here we show that the eIF4E-binding protein, Eap1p, is required for efficient degradation of Vts1p target transcripts and that this role involves the ability of Eap1p to interact with eIF4E. Eap1p does not stimulate deadenylation of Vts1p target transcripts but is instead involved in decapping. Eap1p interacts with Vts1p and mediates an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby the interaction of Vts1p with Eap1p at target mRNAs stimulates decapping.

Sequence-specific RNA binding proteins can add another level of control to the regulation of mRNA stability [7]. Typically these proteins bind mRNA target sequences and interact with other trans factors that influence the rate of mRNA decay. The Smaug (Smg) family of post-transcriptional regulators, which are conserved from yeast to humans, bind RNA through a conserved sterile alpha motif (SAM) domain that interacts with stem-loop structures termed Smg recognition elements (SREs) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Vts1p, the Smg family member in S. cerevisiae, stimulates mRNA degradation through deadenylation by the Ccr4p-Pop2p-Not deadenylase complex [12], [18]. Following deadenylation Vts1p target transcripts are decapped and then degraded by the 59-to-39 exonuclease Xrn1p [18]. A similar mechanism of deadenylation-dependent mRNA decay is employed by Smg in Drosophila [15], [17], [19]. Both Vts1p and Smg interact with the Ccr4p-Pop2p-Not complex suggesting a model whereby these proteins induce transcript decay by recruiting the deadenylase to target mRNAs. Smg also regulates mRNA translation through a separate mechanism involving an interaction with the eIF4E-binding protein Cup [20]. Cup binds to the mRNA cap binding protein eIF4E through a canonical eIF4E-binding motif (YXXXXLW, where W is a hydrophobic amino acid). Cap-dependent translation initiation involves eIF4E recruiting eIF4G to an mRNA, which indirectly mediates recruitment of the 40S ribosome [21]. eIF4G also interacts with eIF4E through an eIF4E-binding motif and thus recruitment of Cup to an mRNA inhibits translation by blocking the eIF4E/ eIF4G interaction [20], [22].
The role of Cup in Smg function led us to speculate that Vts1p might also regulate target mRNAs through an eIF4E-binding protein. While there is no Cup homolog in yeast, two eIF4Ebinding proteins, Caf20p and Eap1p, have been identified [23], [24], [25]. In addition, global genetic analysis revealed synthetically lethal interactions between Eap1p and two deadenylase components, Ccr4p and Pop2p [26], suggesting a functional relationship, either direct or indirect, among the gene products. This genetic interaction combined with the role of the Ccr4p-Pop2p-Not deadenylase in Vts1p-mediated regulation prompted us to test if Eap1p might function with Vts1p to regulate target mRNAs. Using two different Vts1p target mRNAs we demonstrate that Eap1p is required for efficient Vts1p-mediated transcript degradation. Eap1p does not stimulate deadenylation but is instead required for efficient removal of the 59 cap. In addition, Eap1p-mediated stimulation of transcript decay requires binding to eIF4E. We also find that Eap1p biochemically interacts with Vts1p and is able to mediate an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby the Vts1p/Eap1p/eIF4E complex stimulates transcript decapping.

Eap1p is Required for Efficient Decay of Vts1p Target mRNAs
To assess the role of Eap1p in Vts1p function we first examined the stability of a reporter mRNA which recapitulates Vts1pmediated decay in vivo [12]. The GFP-SRE + reporter encodes green fluorescent protein (GFP) under the control of the inducible galactose promoter and has three SREs in its 39 untranslated region (UTR). A transcriptional pulse-chase approach was used to measure the stability of reporter mRNAs by Northern blot after transcriptional induction by galactose and subsequent repression by the addition of glucose. We previously reported that GFP-SRE + mRNA is rapidly degraded in wild-type cells while it is stabilized in a vts1D strain [18]. Here we show that rapid degradation of GFP-SRE + mRNA was compromised in eap1D cells ( Figure 1A). The fact that the GFP-SRE + mRNA was stabilized more in a vts1D strain than in an eap1D strain suggests that while Eap1p plays a role in the decay of this mRNA it is not absolutely required for Vts1p function.
These data could suggest that Eap1p functions in the same pathway or a separate pathway to regulate the stability of GFP-SRE + mRNA. To differentiate between these two possibilities we compared GFP-SRE + mRNA in vts1D cells and eap1D vts1D double delete cells ( Figure 1B) and found that this mRNA has the same stability under these different conditions. This suggests that Vts1p and Eap1p function together in the same pathway to degrade GFP-SRE + mRNA.
To further confirm the importance of Eap1p in the degradation of Vts1p target mRNAs we measured the stability of YIR016W mRNA in eap1D cells, having previously shown that Vts1p binds to this mRNA and regulates its stability through deadenylation, decapping and 59-to-39 exonucleolytic decay [12], [18]. To do this we used a reporter construct in which GFP is fused to the YIR016W ORF under the control of the GAL1 promoter (GFP-YIR016W). This construct allows us to perform transcriptional pulse/chase experiments similar to those described for the GFP-SRE + reporter and the GFP tag allows us to specifically detect this transcript in cells that contain endogenous YIR016W mRNA. We induced GFP-YIR016W reporter transcription by adding galactose to eap1D cells and then shut off transcription with glucose. Similar to our findings using the GFP-SRE + reporter, we found that the stability of GFP-YIR016W mRNA was increased in the eap1D strain as compared to wild-type ( Figure 2). Taken together these data indicate that Eap1p is required for the rapid decay of Vts1p target mRNAs.
The role of Eap1p in the degradation of Vts1p target mRNAs could indicate a general role in the degradation of mRNAs. Alternatively, its role could be more specific, perhaps reflecting a direct function in Vts1p-mediated decay. To explore these possibilities we assessed the stability of a GFP reporter mRNA (GFP-SRE -) which is identical to the GFP-SRE + reporter with the exception that it carries SREs in which the loop sequences are mutated to block Vts1p binding [12] and as such this mRNA is not destabilzed by Vts1p (Figure 3). Transcriptional pulse-chase experiments demonstrated that GFP-SRE -mRNA was not stabilized in eap1D cells and, in fact, the earlier time points suggest a modest destabilization of the mRNA in these cells ( Figure 3). Similar to Vts1p target mRNAs [18], the GFP-SRE -mRNA was destabilized through the major mRNA decay pathway as degradation required Ccr4p (the catalytic subunit of the Ccr4p-Pop2p-Not deadenylase) and the 59-to-39 exonuclease Xrn1p ( Figure S1). Thus, the differential role of Eap1p in the stability of GFP-SRE + and GFP-SRE -mRNAs is consistent with a direct role for Eap1p in the degradation of Vts1p target mRNAs as opposed to a general role in transcript degradation.
Interestingly, these experiments demonstrated that GFP-SRE -mRNA was less stable in a vts1D strain compared to wild-type cells ( Figure 3). We suggest that the physical interaction between Vts1p and the Ccr4p-Pop2p-Not deadenylase complex [18] in wild-type cells sequesters some fraction of the deadenylase into a pool that is unable to act on mRNAs that are not targeted by Vts1p. In a vts1D Figure 1. Eap1p and Vts1p function in the same pathway to destabilize GFP-SRE + mRNA. GFP-SRE + mRNA expression was induced in the indicated strains and then shut-off with glucose and reporter mRNA levels were assayed at the times indicated after transcriptional shutoff by Northern blot. The results of at least three independent experiments were quantitated and normalized using the levels of SCR1 RNA and graphed with error bars representing standard deviation. *Note that an accurate measure of the half-life of GFP-SRE + mRNA can be found in Figure S2. doi:10.1371/journal.pone.0047121.g001 strain this pool is no longer sequestered and as such is free to act on the GFP-SRE -mRNA, thereby accelerating its deadenylation and subsequent degradation. Consistent with this highly speculative model we see a similar stability for GFP-SRE + and GFP-SRE -mRNAs in vts1Dcells in transcriptional pulse-chase experiments ( Figure S2).

Eap1p is not Required for Vts1p-mediated Deadenylation
We next set out to identify at which step in the mRNA decay process Eap1p functions. Given that Vts1p triggers mRNA decay via poly(A) tail shortening, mediated by the Ccr4p-Pop2p-Not deadenylase complex, we analyzed the poly(A) tail length of GFP-SRE + mRNA in eap1D cells to determine whether Eap1p was required for deadenylation of Vts1p target mRNAs. We used an RNase H cleavage method to measure the poly(A) tail length of the GFP-SRE + mRNA over an 8-min transcriptional pulse-chase time course. Total RNA samples from each time point were treated with RNase H in the presence of a specific antisense oligonucleotide that hybridized to the GFP open reading frame. After RNase H cleavage, the poly(A) tail of the resulting short 39-end fragment of the GFP reporter mRNA was measured by high resolution polyacrylamide electrophoresis and Northern blotting. To one sample specific oligonucleotide and oligo(dT) were added and, as such, treatment with RNase H yielded a 39-end fragment lacking a poly(A) tail, which served as a marker for the deadenylated 39end fragment.
Comparison of the RNase H cleavage data from wild-type cells and eap1D cells ( Figure 4) indicate that Eap1p is not required for deadenylation of the GFP-SRE + mRNA. For example, wild-type cells at time zero showed two populations of GFP-SRE + mRNA. One population had tail lengths from 30 to 50 nucleotides while the other had very short tails presumably representing transcripts  that were deadenylated during the 16-min induction with galactose indicating the transcript was being rapidly deadenylated. At time zero eap1D cells also showed these same two populations of GFP-SRE + mRNA, again suggesting that this mRNA is rapidly deadenylated. In fact, eap1D cells show an increase in the fraction of deadenylated GFP-SRE + mRNA suggesting that the mRNA is being more rapidly deadenylated during the 16 minute galactose induction. In addition, in both wild-type and eap1D cells soon after the addition of glucose we saw a decrease in the amount of longer poly(A) tail species and an increase in the amount of the shorter tail population again indicating no deadenylation defect in eap1D cells. At later time points deadenylated species persisted in the eap1D cells, similar to results we reported for mutants that are wildtype for deadenylation but defective for downstream steps such as decapping and 59-to-39 exonucleolytic decay [18]. Taken together these data suggest that Eap1p is not required for Vts1p-mediated deadenylation and if anything it might antagonize this process. Thus, the stabilization of GFP-SRE + mRNA in eap1D cells does not result from a defect in deadenylation but instead must reflect a role for Eap1p in a subsequent step of the decay process.

Eap1p Stimulates mRNA Decapping
After deadenylation Vts1p target transcripts are decapped and then degraded in a 59-to-39 direction by the exonuclease Xrn1p [18]. To determine if Eap1p is involved in the decapping of Vts1p target transcripts we made use of an antibody that recognizes the mRNA 59 cap structure. A transcriptional pulse-chase experiment was performed and RNA harvested 16-min post glucose addition was immunoprecipitated with the 59 cap antibody. The levels of GFP-SRE + mRNA in the immunoprecipitates and in the flow through were assayed via Northern blot to provide a quantitative measure of the percentage of capped versus uncapped mRNA. The results from eap1D cells were compared to results obtained from cells depleted of Dcp2p, which is the catalytic subunit of the decapping complex, and xrn1D cells, which are defective for 59-to-39 exonucleolytic decay. Depletion of Dcp2p was achieved using a strain expressing DCP2 under a tetracycline regulatable promoter (TetO 7 -DCP2), where inhibition of DCP2 transcription results from addition of the tetracycline analog doxycycline [27].
In these experiments we recovered on average ,50% of the GFP-SRE + mRNA. When cells were depleted of Dcp2p and therefore defective for decapping, GFP-SRE + mRNA was enriched 2.4 fold in the immunoprecipitated material, with 36.6% of the total GFP-SRE + mRNA present in the immunoprecipitated fraction and 15.2% present in the supernatant ( Figure 5). In contrast, GFP-SRE + mRNA was not enriched in the immunoprecipitated material from xrn1D cells, with similar levels of GFP-SRE + mRNA found in immunoprecipitated and supernatant fractions. Control experiments confirm the specificity of the antibody for the 59 cap structure as #3.5% of the total SCR1 RNA, which is an RNA polymerase III transcript that lacks a cap structure [28], was precipitated in any experiment. In eap1D cells, GFP-SRE + mRNA was enriched 3 fold in immunoprecipitated material, with 42% found in the immunoprecipitated fraction and 14% in the supernatant. The similar enrichment of GFP-SRE + mRNA in Dcp2p depleted cells and eap1D cells indicates that Eap1p is required for efficient decapping of GFP-SRE + mRNA.

Eap1p Mediates an Interaction between Vts1p and eIF4E
Given the requirement of Eap1p for Vts1p-mediated mRNA decay, we sought to determine whether Vts1p physically interacts with Eap1p in vivo. We performed co-immunoprecipitation experiments using whole-cell lysates from cells expressing Flagtagged Vts1p and HA-tagged Eap1p. Vts1p-Flag was immunoprecipitated using anti-Flag resin, and the immunoprecipitates were analyzed by Western blot. Eap1p-HA was present in the anti-Flag immunoprecipitate when the Vts1p-Flag protein was present ( Figure 6A). As a control, Eap1p-HA was not immunoprecipitated from an extract lacking Vts1p-Flag. The co-immunoprecipitation of Vts1p with Eap1p was RNA independent, as it was observed when Vts1p harbored an amino acid change (A498Q) that blocks its ability to bind RNA (Vts1pRBD 2 ) [12].
The interaction of Vts1p with the eIF4E-binding protein Eap1p led us to hypothesize that Eap1p may mediate an interaction between Vts1p and eIF4E. To test whether Vts1p interacts with eIF4E via Eap1p we repeated our co-immunoprecipitation experiments using whole cell lysates derived from eap1D cells. Consistent with data presented above, Eap1p-HA was present in the anti-Flag immunoprecipitate when the Vts1p-Flag protein was present ( Figure 6B). eIF4E was also detected in Vts1p-Flag immunoprecipitates only in the presence of Eap1p. eIF4E was not significantly enriched when Vts1p-Flag was immunoprecipitated from lysate expressing Eap1p that harbored amino acid changes (Y109A;L114A) in the eIF4E-binding motif that block its ability to bind eIF4E (Eap1p mt ) [29]. In addition, we did not detect a significant interaction between Eap1p mt -HA and Vts1p-Flag, suggesting that binding of Eap1p to eIF4E may stabilize the Vts1p/Eap1p interaction. Taken together these data indicate that Eap1p mediates an interaction between Vts1p and eIF4E through its eIF4E-binding motif.

Binding to eIF4E is Required for Eap1p to Stimulate Transcript Decay
To 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 ability (Eap1p mt -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 Eap1p mt -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 Eap1p mt -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.

Discussion
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 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).  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 vts1D strain ( [12], 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 + and GFP-SRE -mRNAs in wild-type, vts1D and eap1D 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 [30]. 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 [31], [32]. For example, disruption of eIF4G function in yeast results in increased rates of decapping [31]. 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 59 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 [33]. 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 Vts1pmediated decay of the GFP-SRE + mRNA does not involve Dhh1p [18].

The Function of Eap1p in Yeast
Eap1p functions in a variety of biological processes including pseudohyphal growth [29], the response to oxidative stress induced by exposure to diamide and cadmium [34], the response to vesicular transport defects [35] and the regulation of rapamycin-induced inactivation of TOR-signaling [25], [36]. In addition, polysome analysis has shown that Eap1p regulates the translation of specific target transcripts [29]. 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 [30] as well as Puf5 (Aaron Goldstrohm, University of Michigan, personal communication), at least in part, controls which transcripts Eap1p regulates. Figure 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 (Eap1p mt -HA) and 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 Eap1p mt -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 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 [37]. Originally identified as being responsible for transporting eIF4E into the nucleus [38], subsequent experiments showed that depletion of 4E-T from HeLa cells resulted in significant stabilization of A/U rich element (ARE)containing mRNAs [37]. 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 Pbodies, which are sites of mRNA storage and decay [37], [39]. 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.

Yeast Strains and Media
Yeast strains used in this study were derivatives of the wild-type BY4741 [40]. All deletion strains are as described by Winzeler et al., (1999), with the exception of the eap1D strain which was generated for this study by directed PCR-mediated gene deletion (MATa eap1D::KANMX6 leu2D0 his3D1 ura3D0 met15D0). The TetO 7 -DCP2 strain is described in Mnaimneh et al., (2004). The strains were transformed by standard techniques, and plasmids were maintained by growth in selective media.

mRNA Analysis
Transcriptional pulse-chase experiments employed GFP-SRE + , GFP-SRE -, and GFP-YIR016W reporters described in Rendl et al., [18]. For these experiments cells were grown in selective medium containing 2% raffinose at 30uC to mid-log phase and then cooled to 20uC. Due to the short half-life of the GFP-SRE + reporter in wild-type cells, an accurate measure of reporter mRNA stability at 30uC was not possible. Cooling cells to 20uC slowed the decay of GFP-SRE + mRNA to allow for measurement of reporter mRNA half-lives. GFP reporter transcription was initiated by the addition of galactose to a final concentration of 2% for 16 min and then repressed by the addition of glucose to a final concentration of 4%. Total RNA was isolated by glass bead lysis in LET buffer (100 mM LiCl, 20 mM EDTA, 25 mM Tris-HCl at pH 8.0) and LET-equilibrated phenol at the indicated time points and analyzed by Northern blot. SCR1 or ACT1 RNA was used for normalization of transcript levels as indicated.
RNase H cleavage assays were performed as described by Decker and Parker (1993), using an oligonucleotide that hybridized ,330 nt upstream of the GFP reporter poly(A) site. To control for differences in RNA concentration a portion of each RNA sample was analyzed via a standard Northern blot and probed for SCR1 RNA. Northern blots were exposed to PhosphorImager screens and analyzed with ImageQuant Software.
For RNA immunoprecipitations total RNA was isolated from cells expressing the GFP-SRE + reporter during transcriptional pulse-chase experiments 16 min after transcriptional shut-off with glucose. Immunoprecipitations were performed as described in Muhlrad et al. (1994) using monoclonal anti-m 3 G-/m 7 G-cap (Synaptic Systems) with the following modifications. Anti-m 3 G-/ m 7 G-cap antibodies were prebound at 4uC to protein G-Agarose (Roche) in IPPH (500 mM NaCl, 10 mM Tris-Cl at pH 7.5, 0.1% NP-40) for 2 h. The beads were extensively washed in IPPL (150 mM NaCl, 10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 0.05% NP-40) and then incubated at 4uC with 5 mg total RNA in IPPL supplemented with 1 mM DTT and 0.1 U/mL RNasin (Promega) for 2 h. The supernatant was recovered and incubated for 2 h at 4uC a second time with antibody prebound to beads. The pellets were washed twice with IPPL and combined. Bound and unbound mRNA was then isolated from the pellet and supernatant fractions and analyzed by Northern blot.
For Figure 7 transcriptional pulse-chase experiments were performed to analyze reporter mRNA stability as described above. For analysis of protein expression a sample of cells at time 0 were denatured by trichloroacetic acid (TCA) precipitation, lysed with glass beads and the resulting extracts fractionated on SDS-PAGE and analyzed by Western blot using HA antibodies (Santa Cruz) and PSTAIR antibodies (Sigma) as indicated.

Immunoprecipitations
Immunoprecipitation was performed essentially as described previously in Rendl et al. (2008). The plasmid expressing Vts1p-Flag was generated in pRS315 with a C-terminal Flag tag and 574 bases of genomic sequence upstream and 396 bases downstream of the VTS1 ORF. The plasmid expressing Vts1pRBD -Flag was generated in pRS315 and is identical in sequence to Vts1p-Flag with the exception of the A498Q amino acid change. All Vts1p constructs also harbor VSV epitopes that were used for protein detection by Western blot. The Eap1p-HA plasmid was created in pRS316 with a C-terminal 3X HA tag and 490 bases of genomic sequence upstream and 492 bases downstream of the EAP1 ORF. The Eap1p mt -HA construct was generated in pRS316 and is identical in sequence to Eap1p-HA with the exception of the Y109A;L114A amino acid changes. Preparation of protein extracts by glass bead lysis and subsequent immunoprecipitation experiments were preformed as previously described in Rendl et al. (2008). Briefly anti-Flag M2 affinity gel (Sigma) was used to immunoprecipitate Flag-tagged proteins for 3 h at 4uC. After extensive washing Vts1p-Flag and associated proteins were eluted with Flag-peptide (Sigma). VSV antibodies (Bethyl Laboratories), HA antibodies (Santa Cruz) and eIF4E antibodies (a generous gift from N. Sonenberg [25]) were used to detect eluted proteins by Western Blot. Figure S1 GFP-SRE -mRNA is degraded through a Ccr4p/Xrn1p-dependent pathway. GFP-SREgene transcription was induced in wild-type, ccr4D and xrn1D cells with galactose and then shut off with glucose and reporter mRNA levels were assayed at the times indicated after transcriptional shutoff by Northern blot. The results of at least two independent experiments were quantitated and normalized using the levels of SCR1 RNA and graphed with error bars representing standard deviation. Note that there is signficantly less GFP-SRE -mRNA in wild-type cells compared to either ccr4D or xrn1D cells at the 30 minute time point (P,0.03). (TIF) Figure S2 GFP-SRE + and GFP-SRE -mRNAs have the same stability in vts1D cells. GFP-SRE + and GFP-SREgene transcription was induced in vts1D cells with galactose and then shut off with glucose and reporter mRNA levels were assayed at the times indicated after transcriptional shutoff by Northern blot. The results of at least two independent experiments were quantitated and normalized using the levels of SCR1 RNA and graphed with error bars representing standard deviation. Data for GFP-SREis from Figure 3. (TIF)