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Vol. 18, Issue 6, 2274-2287, June 2007
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Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, AZ 85721-0106; and *Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal
Submitted March 5, 2007;
Revised March 28, 2007;
Accepted March 30, 2007
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Parker and Song, 2004). Deadenylation can lead to 3' to 5' degradation, but primarily is followed by removal of the 5' end cap structure by the Dcp1p/Dcp2p decapping enzyme and 5' to 3' degradation of the body of the transcript by the exonuclease Xrn1p. Decapping is a key step of this process, because it precedes and permits the degradation of the body of the mRNA and represents the site of multiple control inputs.
The process of mRNA decapping and translation are mechanistically intertwined and appear to compete with each other, at least in yeast (reviewed in Coller and Parker, 2004). For example, decreasing translation initiation by a variety of means increases the rate of mRNA decapping (LaGrandeur and Parker, 1999; Muhlrad and Parker, 1999; Schwartz and Parker, 1999, 2000). Conversely, inhibition of translation elongation leads to a significant decrease in the rate of mRNA decapping (Beelman and Parker, 1994). Moreover, coimmunoprecipitation experiments suggested that before decapping, the mRNA must exit translation and assemble into a translationally repressed messenger ribonucleoprotein (mRNP) complex capable of decapping (Tharun and Parker, 2001). These results indicate that before decapping mRNAs cease translation and assemble an mRNP containing the decapping machinery.
Additional evidence for a discrete population of nontranslating mRNPs has been that nontranslating mRNAs and the decapping machinery accumulate in discrete cytoplasmic foci called P-bodies (also referred as GW182 or Dcp bodies) (Bashkirov et al., 1997; Ingelfinger et al., 2002; Lykke-Andersen, 2002; van Dijk et al., 2002; Sheth and Parker, 2003; Cougot et al., 2004). P-bodies have now been observed in yeast, insect cells, nematodes, and mammalian cells and contain various proteins implicated in mRNA degradation, including the decapping enzyme (Dcp1p/Dcp2p), activators of decapping Dhh1p, Pat1p, Lsm1-7p, Edc3p, and the exonuclease Xrn1p (reviewed in Anderson and Kedersha, 2006; Eulalio et al., 2007; Sheth and Parker, 2007). Consistent with the reciprocal relationship between mRNA translation and degradation, the assembly of P-bodies is in a dynamic competition with translation (Teixeira et al., 2005; Sheth and Parker, 2007). Moreover, the mRNPs that accumulate in P-bodies have been suggested to be functionally involved in mRNA decapping (Sheth and Parker, 2003; Cougot et al., 2004), mRNA storage (Brengues et al., 2005; Bhattacharyya et al., 2006), general translation repression (Holmes et al., 2004; Coller and Parker, 2005), miRNA-mediated repression (Jakymiw et al., 2005; Liu et al., 2005; Pillai et al., 2005), nonsense-mediated decay (Unterholzner and Izaurralde, 2004; Sheth and Parker, 2006), and possibly viral packaging (Beliakova-Bethell et al., 2006). The existence of P-bodies as a discrete cytoplasmic compartment containing nontranslating mRNAs that can be either degraded or stored suggests that understanding the nature of the protein–protein and protein–RNA interactions that allow the assembly of both mRNPs containing the decapping machinery, as well as larger P-bodies visible by light microscopy, will be important in understanding the control of mRNA translation and degradation.
Two aspects of the assembly of P-bodies have emerged. First, it has been shown that P-bodies require mRNA for their formation and integrity (Teixeira et al., 2005). Second, coimmunoprecipitation and two-hybrid experiments have revealed a dense network of interactions between the components of the mRNA decapping and degradation machinery found in P-bodies (e.g., Hata et al., 1998; Coller et al., 2001; Ho et al., 2002; Fenger-Gron et al., 2005; Gavin et al., 2006; Krogan et al., 2006). However, the specific interactions that mediate the assembly of the translation repression and decapping machinery on mRNAs as well as the aggregation of individual mRNPs into larger P-bodies are largely unknown.
Several proteins have been described as affecting P-body assembly in yeast and mammalian cells. However, the competition between translation and P-body formation suggests that lack of a specific protein can affect P-body formation by either reducing the pool of nontranslating mRNAs, or by decreasing the aggregation of the nontranslating "P-body" mRNPs. For example, in mammalian cells, P-bodies are greatly reduced by knockdown of GW182, RCK/p54, RAP55, miRNA biogenesis in general, LSm4, Hedls/Ge-1, or 4E-T (Andrei et al., 2005; Ferraiuolo et al., 2005; Jakymiw et al., 2005; Pauley et al., 2006). However, for at least LSm4 knockdowns, P-bodies are restored when translation initiation is inhibited by arsenite (Kedersha et al., 2005). Additionally, depletion of GW182 relieves translational repression by miRNAs (Jakymiw et al., 2005; Liu et al., 2005a; Meister et al., 2005; Rehwinkel et al., 2005; Behm-Ansmant et al., 2006). Moreover, 4-ET and RCK/p54 are known to function in translational repression (Andrei et al., 2005; Ferraiuolo et al., 2005). These observations argues that LSm4p, and possibly these other factors as well, are not required for P-body assembly per se, but instead contribute to P-body formation in mammalian cells by increasing the pool of translationally repressed mRNAs. Similarly, in yeast, Dhh1p and Pat1p are required for global translation repression of mRNAs, targeting mRNAs for decapping, and promoting their assembly into P-bodies (Coller and Parker, 2005). Strains lacking Dhh1p and Pat1p are defective in translation repression in response to glucose deprivation and amino acid starvation, mRNA decapping, and consequently in P-body formation (Holmes et al., 2004; Coller and Parker, 2005). In contrast, overexpression of Dhh1p or Pat1p results in inhibition of translation and induces P-body formation (Coller and Parker, 2005).
Given the limited understanding of how P-bodies assemble our goal in this work was to determine the requirement for numerous yeast proteins in P-body assembly and organization. To this end, we examined P-body formation and composition in strains defective in proteins known to accumulate in P-bodies. These experiments revealed specific dependencies in P-body assembly for individual proteins. In addition, this work argues that P-body assembly is redundant and no single known component of P-bodies is absolutely required for P-body assembly although Dcp2p and Pat1p can affect P-body assembly. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Proteins were C terminal tagged with green fluorescent protein (GFP) following the PCR-based gene modification method described by Longtine et al. (1998). These fusion proteins include the full-length protein and are all at least partially functional (Sheth and Parker, 2003). Yeast crosses were carried out using standard laboratory procedures. Strains were grown on standard yeast extract/peptone medium (YP) supplemented with 2% Dextrose (Glu) as carbon source. Strains were grown at 30°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Each strain was then first examined microscopically for accumulation of each protein in P-bodies in yeast grown in glucose-supplemented rich medium during the midlog growth phase, wherein P-bodies are small in wild-type cells (Teixeira et al., 2005). This provides an ideal condition to identify lesions that increase the size of P-bodies. In some cases, strains were also observed under the stress condition of glucose deprivation, which decreases translation initiation and leads to the accumulation of large P-bodies (Ashe et al., 2000; Teixeira et al., 2005). Because P-bodies are large in wild-type cells undergoing a response to glucose deprivation, this provides an opportunity to identify lesions that reduce or compromise P-body assembly. Finally, where relevant we examined the accumulation of some proteins in strains lacking two proteins. The important results are discussed below.
Defects in the Catalysis of Decapping or 5' to 3' Exonuclease Digestion Lead to the Accumulation of P-Bodies
Several observations demonstrated that blocking the catalytic events of decapping or 5' to 3' exonuclease digestion led to increased P-bodies. For example, strains lacking the 5' to 3' exonuclease Xrn1p, accumulated Dhh1p, Pat1p, Lsm1p, Dcp1p, Dcp2p, and Edc3p in P-bodies even in midlog cultures where yeast P-bodies are generally small (Figure 1B, III–VIII). Similarly, during midlog growth the dcp1
strain showed high levels of accumulation of Dhh1p, Pat1p, Lsm1p, Dcp2p, Edc3p, and Xrn1p in P-bodies (Figure 1C, III–IX). These large P-bodies observed in xrn1 or dcp1 strains did not increase significantly when cells were subjected to glucose deprivation, by shifting the cells for 10 min to medium lacking glucose (data not shown and Figure 2B), which is consistent with the observation that xrn1 and dcp1 strains are unable to repress translation during glucose deprivation (Holmes et al., 2004; Coller and Parker, 2005).
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blocks 5' to 3' degradation and the dcp1 blocks decapping, these observations indicate that defects in the catalytic steps of decapping or 5' to 3' degradation lead to increased P-bodies. Moreover, because all the proteins examined accumulated in P-bodies under these conditions, it argues that no other known component of P-bodies is strictly dependent on Xrn1p or Dcp1p for accumulation in P-bodies. This is consistent and extends earlier work showing an accumulation of Dhh1p in P-bodies in xrn1 or dcp1 strains (Sheth and Parker, 2003). Moreover, these observations are consistent with the observation that RNAi-mediated knockdown of the decapping enzyme Dcp2p in mammalian cells leads to an accumulation of P-bodies as assessed by the human ortholog of Dhh1p, RCK/p54, as well as hCcr4p, and hLSm1p (Andrei et al., 2005).
Interestingly, Ccr4p and Pop2p were also clearly observed to accumulate in P-bodies in the xrn1, dcp1, and dcp2 mutant strains (Figure 1, B, I and II, C, I and II, and D, I and II). Similarly, we observed that Ccr4p and Pop2p accumulated at low levels in P-bodies during glucose deprivation (see below, Figure 8). This indicates that Ccr4p and Pop2p can accumulate in yeast P-bodies. This is consistent with earlier observations that other components of the Ccr4p/Pop2p/Not complex, the Not1-5 proteins, are detected in P-bodies in dcp1 strains (Muhlrad and Parker, 2005) and that yeast Ccr4p can be observed in small foci during a water stress response (Sheth and Parker, 2003). In addition, mammalian and Drosophila Ccr4p also accumulate in P-bodies (Cougot et al., 2004; Temme et al., 2004; Andrei et al., 2005). These results indicate that the Ccr4p/Pop2p/Not1-5p complex accumulates in P-bodies in yeast as well as other eukaryotes. However, the limited amount of Ccr4p-GFP and Pop2p-GFP seen in P-bodies compared with other factors suggests that these proteins are present at reduced stoichiometry.
Dcp2p Is Required for Dcp1p Accumulation in P-Bodies
Strains lacking Dcp2p also showed increased P-bodies in midlog cultures with accumulations of Ccr4p, Pop2p, Dhh1p, Pat1p, Lsm1p, Edc3p, and Xrn1p in P-bodies (Figure 1D, I–V and VIII–IX), but with some differences from dcp1 or xrn1 strains. First, despite the increased P-bodies in dcp2 strains as judged by other protein components, Dcp1p-GFP was distributed throughout the cell and completely absent from P-bodies (Figure 1D, VI). Moreover, Western analysis showed that Dcp1p-GFP was still efficiently expressed in the dcp2 strain (data not shown). Because dcp1 and dcp2 strains have identical and complete blocks to decapping (Beelman et al., 1996; Dunckley and Parker, 1999) and all other proteins accumulate in P-bodies in the dcp2 strain, this observation argues that Dcp2p is required to recruit Dcp1p to P-bodies.
Two additional observations confirm Dcp2p is required for Dcp1p recruitment to P-bodies. First, even when dcp2 strains are undergoing a response to glucose deprivation, which increases Dcp1p-GFP accumulation in P-bodies in wild-type strains, Dcp1p-GFP is completely absent from P-bodies in a dcp2 strain (Figure 2A, IV). In contrast, Dcp2p-GFP accumulates in P-bodies independent of Dcp1p with or without glucose deprivation conditions (Figure 2B, II and IV). Moreover, other P-body markers still accumulate in P-bodies in a dcp2 with or without glucose deprivation conditions (Figure 1D and data not shown). Second, although Dcp1p-GFP accumulates in P-bodies to high levels in the xrn1 strain (Figure 2C, I), this accumulation is completely lost in the xrn1 dcp2 double mutant strain (Figure 2C, II). The requirement of Dcp2p for Dcp1p recruitment to P-bodies is consistent with the direct physical interaction between Dcp1p and Dcp2p in yeast (Steiger et al., 2003; She et al., 2004, 2006) and with the observation that specific point mutations in residues of Dcp2p that disrupt the physical interaction between Dcp2p and Dcp1p prevent Dcp1p from accumulating in P-bodies (Decker and Parker, unpublished observation). These results indicate that Dcp1p is recruited to the P-body through interactions with Dcp2p.
Dcp2p Has an Additional Role in P-Body Assembly or Persistence
A second interesting result observed with the dcp2 strain was that P-bodies were reproducibly smaller than in dcp1 or xrn1 strains (Figure 1, B–D). This can be easily seen by comparing the accumulation of Dhh1p, Pat1p, Lsm1p, or Xrn1p in dcp1 strains compared with dcp2 strains (compare Figure 1C, III–V and IX with 1D, III–V and IX). Because both dcp1 and dcp2 strains show absolute blocks to decapping (Beelman et al., 1996; Dunckley and Parker, 1999), this difference in P-body size must reflect a difference in the function of Dcp1p or Dcp2p in the formation of P-bodies independent of their catalytic role in decapping. Interestingly, the smaller size of P-bodies in the dcp2 strain relative to the dcp1 strain was not as pronounced when Edc3p was examined (Figure 1D, VIII), suggesting some difference in how Edc3p's accumulation in P-bodies is affected compared with other factors.
Two possible models can explain the difference between P-body size in the dcp1 and dcp2 strains, which we distinguished by the analysis of a dcp1 dcp2 double mutant. First, it could be that Dcp1p is also an inhibitor of P-body formation and that P-bodies are larger in the dcp1 strain compared with the dcp2 strain because of the loss of the Dcp1p inhibitory role on P-body formation. In this model, a dcp1 dcp2 double mutant is predicted to have large P-bodies similar to the dcp1 strain. Alternatively, Dcp2p might have an additional role in the assembly or maintenance of P-bodies, possibly through additional protein–protein interactions. In this model, a dcp1 dcp2 strain is predicted to be phenotypically similar to a dcp2 strain.
To distinguish these models, we examined the accumulation of Dhh1p and Pat1p in dcp1 dcp2 double mutants strains as compared with dcp1 and dcp2 single mutant strains. Strikingly, we observed that both Dhh1p-GFP and Pat1p-GFP accumulated in P-bodies in the dcp1 dcp2 double mutant to the same extent seen in the dcp2 single mutant (Figure 3, V and VI). This provides evidence that Dcp2p has some role in the assembly or maintenance of P-bodies. A likely possibility is that the multiple interactions that Dcp2p has with other components of P-bodies contribute to the assembly of the P-body mRNP or to the interactions that allow P-body aggregation into visible structures in the light microscope.
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led to a small, but clear, decrease in the accumulation of Dcp2p-GFP and Edc3p-GFP in P-bodies in midlog cultures (Figure 4B, V and VI). Furthermore, we observed pop2 led to a small decrease in the accumulation Dcp1p-GFP and Dcp2p-GFP in P-bodies (Figure 4C, IV and V). This is consistent with the ccr4 and pop2 affecting deadenylation (Tucker et al., 2001) and previous work arguing that in midlog growth phase deadenylated mRNAs preferentially associate with P-body components such as Lsm1p (Tharun and Parker, 2001). In midlog cultures, the ccr4 or pop2 strains did not show a significant difference in the concentration of Dhh1p-GFP, Pat1p-GFP, Lsm1p-GFP, or Xrn1p-GFP in P-bodies (Figures 4B, I–III and VII, and 4C, I-III and VII). However, because it is difficult to observe these proteins in P-bodies in midlog growth, we are unable to determine if the ccr4 or pop2 affects their accumulation in P-bodies under these conditions.
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strains in response to glucose deprivation stress, where decreases in P-bodies are more easily identified, Pat1p-GFP and Edc3p-GFP accumulated in P-bodies in a manner similar to wild-type cells (Figure 4B, IX and XIII), whereas Dhh1p-GFP, Lsm1-GFP, Dcp1p-GFP, Dcp2p-GFP, and Xrn1p-GFP accumulated but less pronouncedly compared with wild-type cells (Figure 4B, VIII, X–XII, and XIV). This slightly reduced accumulation of Dhh1p in P-bodies in these mutant may be due to the reduced levels of protein expression in ccr4 strains, as has been shown to be the case for Dhh1p (Sheth and Parker, 2003). In contrast, after glucose deprivation in a pop2 strain, Dhh1p, Pat1p, Lsm1p, Dcp1p, Dcp2p, Edc3p, and Xrn1p accumulated in P-bodies in a manner similar to wild-type cells (Figure 4C, VIII–XIV). Taken generally, these results indicate that during glucose deprivation, neither Ccr4p nor Pop2p has a large impact on P-body assembly. This is consistent with recent results indicating that poly(A)+ mRNAs are translationally repressed during glucose deprivation and accumulate in P-bodies (Brengues and Parker, in press).
Dhh1p, Pat1p, and Lsm1p Have Different Effects on P-Body Assembly and Composition
The proteins Dhh1p, Pat1p, and Lsm1p are all found in P-bodies and function as general decapping activators (reviewed in Coller and Parker, 2004). To determine the roles of these proteins on P-body assembly we examined the accumulation of GFP tagged version of Dcp1p, Dcp2p, Edc3p, Xrn1p, Dhh1p, Pat1p, and Lsm1p in lsm1, pat1 or dhh1 strains. Because results from mammalian cells indicate that the Lsm1-7p complex must be intact to localize to P-bodies (Ingelfinger et al., 2002), and lsm1 is sufficient to fully inactivate this complex with respect to decapping (Tharun et al., 2000), the lsm1 should be sufficient to address the contribution of the entire Lsm1p-7p complex to P-body formation. In addition, we examined both the affect of the deletions on P-bodies during midlog growth, where P-bodies are small and increases are easily seen, and during glucose deprivation, where P-bodies are large, and decreases in P-body assembly are more easily observed. These results are presented in Figure 5 and important observations are discussed below.
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strains showed increased accumulation of Dcp1p, Dcp2p, Edc3p, Xrn1p, and Dhh1p in P-bodies even in midlog growth cells (Figure 5B, I–V). Pat1p did not accumulate in P-bodies in midlog cultures in the lsm1 strain, perhaps because of an effect of the lsm1 on the nuclear-cytoplasmic distribution of Pat1p (Figure 5B, VI; see below). The accumulation of all P-body components except Pat1p in P-bodies in lsm1 strain is consistent with the decapping defect seen in lsm1 strains (Boeck et al., 1998; Bouveret et al., 2000; Tharun et al., 2000). Moreover, this observation argues that the rate-limiting step in decapping in lsm1 strains is after mRNAs assembly into an mRNP that can accumulate in P-bodies. This identifies an important function of the Lsm1-7p complex in triggering decapping after translation repression and accumulation of the mRNA in the P-body mRNP. Consistent with the partial block to mRNA decapping in the lsm1 strain, the accumulation of P-bodies is not as strong in the lsm1 strain compared with the dcp1 strain, where decapping is absolutely blocked (compare Figure 1C with 5B). Moreover, in lsm1 strains, P-bodies still increased during glucose deprivation compared with nonstressed lsm1 cells (Figure 5B, VIII–XIII), which suggests that lsm1 strains do not have a maximal accumulation of P-bodies.
In contrast to the accumulation of P-bodies in the lsm1 strain, in general the pat1 and dhh1 strains showed either minor or no reduction of P-bodies during midlog growth, respectively, as assessed by Dcp1p, Dcp2p, Edc3p, and Xrn1p (Figure 5, C, I–IV, and D, I–IV). Interestingly, we did observe a minor increase in P-bodies during midlog growth for dhh1 and pat1 cells in a subpopulation of the cells (Figure 5). This suggests that Dhh1p and/or Pat1p can affect the rate of mRNA decay within P-bodies or the rate of mRNA exit from P-bodies in at least some cells. However, during glucose deprivation the pat1 strain and to a more modest effect the dhh1 strain showed a reduction in the amount of P-bodies formed (compare Figure 5A, VIII–XIV, with 5C, VIII–XIV and 5D, VIII–XIV). This is consistent with earlier results that dhh1 and pat1 strains are partially defective in translation repression during glucose deprivation (Holmes et al., 2004; Coller and Parker, 2005). Because both dhh1 and pat1 appear to affect translation repression similarly during glucose deprivation (Holmes et al., 2004; Coller and Parker, 2005), this suggests Pat1p may have a more significant role in P-body assembly and aggregation than Dhh1p (see below and Discussion).
Pat1p Is Required for Recruitment of Lsm1p to P-Bodies
A second important result from these comparisons was that strains lacking Pat1p failed to accumulate Lsm1p in P-bodies with or without glucose repression (Figure 5C, VII and XIV). This argued that Pat1p is required for Lsm1-7p to be recruited to P-bodies. However, because the loss of Pat1p can affect the size of P-bodies during glucose repression, we verified this result under conditions where P-bodies were large even in a pat1 strain. To do this, we examined whether Lsm1p accumulates in P-bodies in a pat1 strain at high cell density, where P-bodies are large (Teixeira et al., 2005). Consistent with Pat1p being required for Lsm1p to enter into P-bodies, we observed that pat1 cells did not accumulate Lsm1p in P-bodies at high OD (Figure 6A, II). These observations demonstrate that Pat1p is required to recruit the Lsm1-7p complex to P-bodies.
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strain Pat1p accumulated in a large circular region of the cell that was similar in size the nucleus (Figure 5B, VI). Subsequent DNA staining with DAPI revealed that this circular region of Pat1p concentration colocalized with the nucleus (Figure 6B). This observation indicates that Lsm1p, and presumably the whole Lsm1-7p complex, plays a role in decreasing the concentration of Pat1p in the nucleus and increasing the cytoplasmic concentration of Pat1p. These results suggest that Pat1p is a likely nuclear-cytoplasmic shuttling protein. This conclusion is consistent with earlier work showing that Pat1p shows physical interactions with the mRNA export factor Crm1p (Jensen et al., 2000).
Despite the nuclear concentration of Pat1p in the lsm1 mutant, three observations suggest that some Pat1p is still in the cytoplasm and is functioning in decapping. First, even in the lsm1 strain during midlog growth, some Pat1p-GFP was observed distributed in the cytoplasm (Figure 5B, VI). Second, when lsm1 cells were glucose deprived some Pat1p was seen to accumulate in P-bodies (Figure 5B, XIII). Third, when P-bodies accumulate because of dcp1, Pat1p-GFP is still present in P-bodies, even in the absence of Lsm1p, albeit at a reduced level compared with a dcp1 alone (Figure 6C, III). These observations indicate that Lsm1p is not absolutely required for Pat1p to enter P-bodies, although it clearly appears to decrease the amount of Pat1p seen in P-bodies, at least in part by affecting the distribution of Pat1p between the nucleus and the cytoplasm. Interestingly, although P-bodies accumulate due to xrn1, Pat1p-GFP was no longer observed in P-bodies in the xrn1 lsm1 double mutant (Figure 6C, IV). This further suggests the idea that Pat1p/Lsm1-7p complex/Xrn1p form a functionally important complex, which is consistent with their previous biochemical copurification (Bouveret et al., 2000).
Double Mutants Indicate Pat1p Functions Before Lsm1p in mRNA Decapping
The above observation suggest a model for P-body assembly wherein Pat1p acts early in the process and is required to recruit the Lsm1-7p complex, which functions later to enhance decapping rate. A prediction of this model is that a pat1 lsm1 double mutant should show the phenotype of the pat1 single mutant strain and not the lsm1 strain. To test this prediction, we examined the accumulation of GFP tagged Dcp1, Dcp2, Edc3p and Xrn1p in pat1 lsm1 strains. For comparison, we also examined the same proteins in a dhh1 lsm1 strain.
A clear and significant observation was that during midlog growth the pat1 lsm1 strain showed little P-body accumulation of all four proteins similar to a pat1 strain (Figure 7, E–H) and unlike a lsm1 strain where P-bodies are increased (Figure 5B). This result provides additional evidence that Pat1p acts upstream of Lsm1p in the process of P-body assembly and mRNA decapping. In contrast, we observed that dhh1 lsm1 leads to increased concentration of Dcp1p, Dcp2p, Edc3p, and Xrn1p in P-bodies, because P-bodies increased in size and number compared with both wild-type cells and dhh1 or lsm1 single-mutant strains (Figure 7, A–D). This suggests that Dhh1p, like Lsm1p, can also play a role in the actual rate of decapping after assembly of the mRNP capable of aggregation in P-bodies.
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, pat1 or lsm1 strains compared with a wild-type strain, where small but clear accumulation of Ccr4p and Pop2p in P-bodies could be seen during glucose deprivation (Figure 8A, III, IV, VII, VIII, XI, XII, XV, and XVI). This suggests that the accumulation of Ccr4p and Pop2 in P-bodies requires Dhh1p, Pat1p, and Lsm1p. However, a limitation of this analysis is that the accumulation of Ccr4p and Pop2p in P-bodies in the wild-type strain is relatively low; thus it is difficult to make a robust conclusion from this experiment alone.
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, pat1 or lsm1 affected the accumulation of Ccr4p or Pop2p in P-bodies in combination with xrn1 where the accumulation of Ccr4p and Pop2p in P-bodies is easily observed (Figure 1). We observed that the dhh1, pat1 or lsm1 all reduced the accumulation of Ccr4p and Pop2p in P-bodies compared with xrn1 single mutant (Figure 8B, III–VIII). This is consistent with the observations during glucose deprivation and argues that the efficient recruitment of Ccr4p and Pop2p to P-bodies requires Dhh1p, Pat1p, and the Lsm1-7p complex. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One protein that can affect P-body formation was Dcp2p. This was surprising because strains lacking Dcp2p show a complete block to decapping and therefore, like dcp1 strains, would be expected to accumulate very large P-bodies. However, dcp2 strains had reproducibly smaller P-bodies than dcp1 or xrn1 strains, although dcp2 strains showed enhanced P-bodies compared with wild-type strains (Figure 1). Moreover, because dcp2 strains have smaller P-bodies than a dcp1 strain as assessed by seven different proteins, it strongly argues that the difference is in the actual formation of P-bodies themselves and not simply a change in P-body composition. Finally, because dcp2 dcp1 strains are similar to dcp2 single mutant strains alone when assessed for P-body formation, it argues that Dcp2p is required for optimal P-body accumulation (Figure 3). Because Dcp2p shows direct physical interactions with Dcp1p, Dhh1p, Edc3, and possibly Pat1p (Steiger et al., 2003; She et al., 2004; Decker, Pilkington, and Parker, unpublished observation), the simplest possibility is that the multiple protein–protein interactions that Dcp2p participates in either stabilizes the P-body monomer mRNP or helps to provide cross-linking interactions between individual mRNPs, thereby contributing to aggregation of multiple mRNPs into a larger P-body. Thus, Dcp2p is not only the catalytic subunit of the decapping enzyme, but also has a second role in promoting P-body assembly or maintenance.
Several observations argue that Pat1p is likely to affect P-body formation in multiple manners. First, because Pat1p overexpression leads to inhibition of translation (Coller and Parker, 2005), one possible role of Pat1p is likely to be to inhibit translation initiation in some manner, thereby contributing to P-body formation by affecting the size of the pool of nontranslating mRNA, which can then aggregate into P-bodies. Second, because P-bodies are reduced in pat1 strains, even during glucose deprivation, where translation is markedly repressed even in a pat1 strain, it suggests that Pat1p also plays a role in assembly of P-bodies (Figure 5). The role of Pat1p in assembly is again likely to be through multiple protein–protein interactions that promote formation of the P-body monomer mRNP, and cross-linking between individual mRNPs. Finally, we suggest that Pat1p has a final role in promoting mRNA decapping after assembly of the P-body mRNP. This is suggested by both the requirement for Lsm1p for efficient decapping after P-body assembly, and the requirement for Pat1p for assembly of Lsm1p into P-bodies.
Specific Dependencies in P-Body Assembly
Our analysis of yeast P-bodies suggests that there are some clear dependencies in the assembly of specific components. For example, the assembly of Dcp1p in P-bodies is dependent on Dcp2p (Figures 1 and 2). This is consistent with the direct physical interaction between these components from yeast (Steiger et al., 2003; She et al., 2004, 2006).
A second clear dependency is that Pat1p is required for the recruitment of Lsm1p to P-bodies (Figures 5 and 6). Because Lsm1p is a component of the Lsm1-7p complex and Lsm1-7p complex formation is required for its recruitment to P-bodies and for the Lsm1-7 complex to function in mRNA decay (Bouveret et al., 2000; Tharun et al., 2000, 2005; Ingelfinger et al., 2002), this suggests that Pat1p is required for the recruitment of the entire Lsm1-7p complex into P-bodies. This is consistent with the physical interactions between Pat1p and the Lsm1-7p complex (Bouveret et al., 2000; Tharun et al., 2000).
Pat1p Acts Early in Translation Repression and mRNA Decapping While Recruiting the Lsm1-7p Complex To Trigger Decapping in a Late Step
Our results also argue that the Lsm1-7p complex has an important role after P-body assembly in the actual triggering of decapping. The key observation is that lsm1 strains show an accumulation of P-bodies, as judged by the subcellular distribution of Dcp1p, Dcp2p, Edc3p, Xrn1p, and Dhh1p (Figure 5). This is consistent with the observed defect in mRNA decapping in the lsm1 strains (Boeck et al., 1998; Bouveret et al., 2000; Tharun et al., 2000) and indicates that the decapping mRNP can assemble and aggregate in P-bodies in the absence of the Lsm1p, but is then deficient at actual decapping, and hence a pool of mRNAs accumulates in P-bodies. It should be noted that these results do not rule out a potential earlier role of the Lsm1-7p complex in translation repression or assembly of the monomer unit, they just reveal what becomes the rate-limiting step in the absence of Lsm1p.
An important implication of the function of the Lsm1-7p complex in triggering decapping after P-body formation is that this role of the Lsm1-7p complex could be used to alter the fate of mRNAs within P-bodies. Specifically, individual mRNAs that are destined for storage could simply be packaged into an mRNP that is lacking the Lsm1-7p complex and thereby have a reduced rate of mRNA decapping. Similarly, in certain biological contexts where mRNA storage is important, the Lsm1-7p complex may be lacking from P-bodies and thereby converts the fate of mRNAs within P-bodies into storage.
Our data also indicate that Pat1p is required for Lsm1p to assemble into P-bodies. The key observation is that P-bodies formed in pat1 strains lack Lsm1p, even under glucose deprivation (Figure 5) or high OD (Figure 6). This is also consistent with earlier work showing that the coimmunoprecipitation of Lsm1p with Dcp2p is greatly reduced in a pat1 strain (Tharun and Parker, 2001). The simplest model is that Pat1p directly interacts with Lsm1-7p complex and recruits it to P-bodies, although one anticipates that Pat1p might also facilitate the interaction of the Lsm1-7p complex with other components of P-bodies.
The phenotypes of lsm1 and pat1 strains suggest a model for these proteins function wherein Pat1p participates early in translation repression and the movement of mRNAs into P-bodies and the Lsm1-7p complex has a rate limiting role at a later stage to trigger decapping. The early role of Pat1p is suggested by the observations that overexpression of Pat1p inhibits translation (Coller and Parker, 2005) and that pat1 strains show reduced P-bodies even under glucose deprivation conditions (Figure 5). The late role of Lsm1p is indicated by the accumulation of P-bodies in the lsm1 strain. Moreover, epitasis analysis is consistent with Pat1p acting before Lsm1-7p because pat1 lsm1 double mutant strains show reduced P-bodies similar to pat1 strains (Figure 7).
Pat1p Is a Nuclear-Cytoplasmic Shuttling Protein and Its Distribution Is Affected by Lsm1p
Our results indicate that Pat1p is likely to be a nuclear-cytoplasmic shuttling protein and its distribution between the nucleus and the cytoplasm is affected by the Lsm1-7p complex. The key observation is that in lsm1 strains, Pat1p accumulates in the nucleus (Figures 5 and 6). Consistent with Pat1p being a nuclear-cytoplasmic shuttling protein, previous work has identified a two-hybrid interaction between Pat1p and Crm1p, which is involved in mRNA export (Jensen et al., 2000). In addition, Pat1p was identified in a proteomic analysis of the components of the penta-snRNP, which is a large nuclear complex involved in pre-mRNA splicing (Stevens et al., 2002). Interestingly, this nuclear localization is not unique to Pat1p in lsm1 strains, because we also detect a clear accumulation of Dhh1p in the nucleus in lsm1 strains under glucose deprivation conditions (Figure 5).
In principle, the accumulation of Pat1p in the nucleus in lsm1 strain could be explained in two manners. First, it could be that interaction of the Lsm1-7p complex with Pat1p in the nucleus is required for efficient export of Pat1p from the nucleus, perhaps in association with mRNAs. This model would imply that an mRNP structure similar to a P-body mRNP may form on some, or all, mRNAs in the nucleus and be transported with them to the cytoplasm. An alternative, and potentially overlapping, model is that interaction of Pat1p with the Lsm1-7p complex in the cytoplasm limits import of Pat1p into the nucleus. Future experiments should be able to distinguish between these models.
Pat1p might function in the nucleus in one of two manners. First, it could be that Pat1p plays a role in a nuclear process such as splicing or degradation of aberrant mRNAs in the nucleus. Alternatively, Pat1p may enter the nucleus to become assembled into nascent mRNPs before export to the cytoplasm, which might lead to those mRNAs entering the cytoplasm in a translationally repressed mRNP that is directly targeted to P-bodies. Such potential targeting of nascent mRNPs might occur on all mRNAs or might be more specific to subclasses of mRNAs, or biological contexts, where translation repression of nascent transcripts is particularly important. Interestingly, one example of such a context is in the biogenesis of maternal mRNAs, which are exported to the cytoplasm and then stored in maternal germ granules, which are related to P-bodies (Anderson and Kedersha, 2006; Sheth and Parker, 2007). Here it is worth noting that the Xenopus ortholog of Dhh1p is a nuclear-cytoplasmic shuttling protein (Smillie and Sommerville, 2002) and may play a role in packaging nascent mRNAs for direct targeting to storage particles. An interesting area of future work will be to understand the functional significance of P-body components shuttling between the nucleus and the cytoplasm.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Roy Parker (rrparker{at}u.arizona.edu).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Andrei, M. A., Ingelfinger, D., Heintzmann, R., Achsel, T., Rivera-Pomar, R., Luhrmann, R. (2005). A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11, 717–727.
Ashe, M. P., De Long, S. K., Sachs, A. B. (2000). Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell 11, 833–848.
Bashkirov, V. I., Scherthan, H., Solinger, J. A., Buerstedde, J. M., Heyer, W. D. (1997). A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates. J. Cell Biol 136, 761–773.
Beelman, C. A. and Parker, R. (1994). Differential effects of translational inhibition in cis and in trans on the decay of the unstable yeast MFA2 mRNA. J. Biol. Chem 269, 9687–9692.
Beelman, C. A., Stevens, A., Caponigro, G., LaGrandeur, T. E., Hatfield, L., Fortner, D. M., Parker, R. (1996). An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382, 642–646.[CrossRef][Medline]
Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4, NOT deadenylase and DCP1, DCP2 decapping complexes. Genes Dev 20, 141885–98.
Beliakova-Bethell, N., Beckham, C., Giddings, T. H. Jr, Winey, M., Parker, R., Sandmeyer, S. (2006). Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components. RNA 12, 94–101.
Bhattacharyya, S. N., Habermacher, R., Martine, U., Closs, E. I., Filipowicz, W. (2006). Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125, 1111–1124.[CrossRef][Medline]
Boeck, R., Lapeyre, B., Brown, C. E., Sachs, A. B. (1998). Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol. Cell. Biol 18, 5062–5072.
Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M., Seraphin, B. (2000). A Sm-like protein complex that participates in mRNA degradation. EMBO J 19, 1661–1671.[CrossRef][Medline]
Brengues, M. and Parker, R. (2007). Accumulation of polyadenylated mRNA, Pab1p, eIF4E and eIF4G with P-bodies in Saccharomyces cerevisiae. Mol. Biol. Cell (in press).
Brengues, M., Teixeira, D., Parker, R. (2005). Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489.
Coller, J. M., Tucker, M., Sheth, U., Valencia-Sanchez, M. A., Parker, R. (2001). The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7, 1717–1727.[Abstract]
Coller, J. and Parker, R. (2004). Eukaryotic mRNA decapping. Annu. Rev. Biochem 73, 861–890.[CrossRef][Medline]
Coller, J. and Parker, R. (2005). General translational repression by activators of mRNA decapping. Cell 122, 875–886.[CrossRef][Medline]
Cougot, N., Babajko, S., Seraphin, B. (2004). Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol 165, 31–40.
Dunckley, T. and Parker, R. (1999). The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J 18, 5411–5422.[CrossRef][Medline]
Eulalio, A., Behm-Ansmant, I., Izaurralde, E. (2007). P bodies: at the crossroads of post-transcriptional pathways. Nat. Rev. Mol. Cell Biol 8, 19–22.[CrossRef][Medline]
Fenger-Gron, M., Fillman, C., Norrild, B., Lykke-Andersen, J. (2005). Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol. Cell 20, 905–915.[CrossRef][Medline]
Ferraiuolo, M. A., Basak, S., Dostie, J., Murray, E. L., Schoenberg, D. R., Sonenberg, N. (2005). A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J. Cell Biol 170, 913–924.
Gavin, A. C., et al. (2006). Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636.[CrossRef][Medline]
Hata, H., Mitsui, H., Liu, H., Bai, Y., Denis, C. L., Shimizu, Y., Sakai, A. (1998). Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae. Genetics 148, 571–579.
Hatfield, L., Beelman, C. A., Stevens, A., Parker, R. (1996). Mutations in trans-acting factors affecting mRNA decapping in Saccharomyces cerevisiae. Mol. Cell. Biol 16, 5830–5838.[Abstract]
Ho, Y., et al. (2002). Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183.[CrossRef][Medline]
Holmes, L. E., Campbell, S. G., De Long, S. K., Sachs, A. B., Ashe, M. P. (2004). Loss of translational control in yeast compromised for the major mRNA decay pathway. Mol. Cell. Biol 24, 2998–3010.
Ingelfinger, D., Arndt-Jovin, D. J., Luhrmann, R., Achsel, T. (2002). The human Lsm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci. RNA 8, 1489–1501.[Abstract]
Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., Hamel, J. C., Fritzler, M. J., Chan, E. K. (2005). Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol 7, 1267–1274.[Medline]
Jensen, T. H., Neville, M., Rain, J. C., McCarthy, T., Legrain, P., Rosbash, M. (2000). Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling. Mol. Cell. Biol 20, 8047–8058.
Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M. J., Scheuner, D., Kaufman, R. J., Golan, D. E., Anderson, P. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol 169, 871–884.
Krogan, N. J., et al. (2006). Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643.[CrossRef][Medline]
Kshirsagar, M. and Parker, R. (2004). Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae. Genetics 166, 2729–739.
LaGrandeur, T. and Parker, R. (1999). The cis acting sequences responsible for the differential decay of the unstable MFA2 and stable PGK1 transcripts in yeast include the context of the translational start codon. RNA 5, 420–433.[Abstract]
Liu, J., Valencia-Sanchez, M. A., Hannon, G. J., Parker, R. (2005). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol 7, 7719–723.[CrossRef][Medline]
Liu, J., Rivas, F. V., Wohlschlegel, J., Yates, J. R. 3rd, Parker, R., Hannon, G. J. (2005a). A role for the P-body component GW182 in microRNA function. Nat. Cell Biol 7, 121261–1266.[Medline]
Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Lykke-Andersen, J. (2002). Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol. Cell. Biol 22, 8114–8121.
Meister, G., Landthaler, M., Peters, L., Chen, P. Y., Urlaub, H., Luhrmann, R., Tuschl, T. (2005). Identification of novel argonaute-associated proteins. Curr. Biol 15, 232149–2155.[CrossRef][Medline]
Meyer, S., Temme, C., Wahle, E. (2004). Messenger RNA turnover in eukaryotes: pathways and enzymes. Cri. Rev. Biochem. Mol. Biol 39, 197–216.[CrossRef]
Muhlrad, D. and Parker, R. (1999). Recognition of yeast mRNAs as "nonsense containing" leads to both inhibition of mRNA translation and mRNA degradation: implications for the control of mRNA decapping. Mol. Biol. Cell 10, 3971–3978.
Muhlrad, D. and Parker, R. (2005). The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p. EMBO J 24, 1033–1045.[CrossRef][Medline]
Parker, R. and Song, H. (2004). The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol 11, 121–127.[CrossRef][Medline]
Pauley, K. M., Eystathioy, T., Jakymiw, A., Hamel, J. C., Fritzler, M. J., Chan, E. K. (2006). Formation of GW bodies is a consequence of microRNA genesis. EMBO Rep 7, 904–910.[CrossRef][Medline]
Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., Filipowicz, W. (2005). Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573–1576.
Rehwinkel, J., Behm-Ansmant, I., Gatfield, D., Izaurralde, E. (2005). A crucial role for GW182 and the DCP1, DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11, 111640–1647.
Schwartz, D. C. and Parker, R. (1999). Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae. Mol. Cell. Biol 19, 5247–5256.
Schwartz, D. and Parker, R. (2000). mRNA decapping in yeast requires dissociation of the cap binding protein, eukaryotic translation initiation factor 4E. Mol. Cell. Biol 20, 7933–7942.
She, M., Decker, C. J., Sundramurthy, K., Liu, Y., Chen, N., Parker, R., Song, H. (2004). Crystal structure of Dcp1p and its functional implications in mRNA decapping. Nat. Struct. Mol. Biol 11, 3249–256.[CrossRef][Medline]
She, M., Decker, C. J., Chen, N., Tumati, S., Parker, R., Song, H. (2006). Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe. Nat. Struct. Mol. Biol 13, 163–70.[CrossRef][Medline]
Sheth, U. and Parker, R. (2003). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808.
Sheth, U. and Parker, R. (2006). Targeting of aberrant mRNAs to cytoplasmic processing bodies. Cell 125, 61095–1109.[CrossRef][Medline]
Sheth, U. and Parker, R. (2007). P-bodies and the control of mRNA translation and degradation. Mol. Cell 25, 5635–646.[CrossRef][Medline]
Smillie, D. A. and Sommerville, J. (2002). RNA helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of stored mRNP particles. J. Cell Sci 115, 395–407.
Steiger, M., Carr-Schmid, A., Schwartz, D. C., Kiledjian, M., Parker, R. (2003). Analysis of recombinant yeast decapping enzyme. RNA 9, 231–238.
Stevens, S. W., Ryan, D. E., Ge, H. Y., Moore, R. E., Young, M. K., Lee, T. D., Abelson, J. (2002). Composition and functional characterization of the yeast spliceosomal penta-snRNP. Mol. Cell 9, 31–44.[CrossRef][Medline]
Teixeira, D., Sheth, U., Valencia-Sanchez, M. A., Brengues, M., Parker, R. (2005). Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371–382.
Temme, C., Zaessinger, S., Meyer, S., Simonelig, M., Wahle, E. (2004). A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J 23, 142862–2871.[CrossRef][Medline]
Tharun, S., He, W., Mayes, A. E., Lennertz, P., Beggs, J. D., Parker, R. (2000). Yeast Sm-like proteins function in mRNA decapping and decay. Nature 404, 515–518.[CrossRef][Medline]
Tharun, S. and Parker, R. (2001). Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p–7p complex on deadenylated yeast mRNAs. Mol. Cell 8, 1075–1083.[CrossRef][Medline]
Tharun, S., Muhlrad, D., Chowdhury, A., Parker, R. (2005). Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection. Genetics 170, 33–46.
Tucker, M., Valencia-Sanchez, M. A., Staples, R. R., Chen, J., Denis, C. L., Parker, R. (2001). The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104, 377–386.[CrossRef][Medline]
Unterholzner, L. and Izaurralde, E. (2004). SMG7 acts as a molecular link between mRNA surveillance and mRNA decay. Mol. Cell 16, 4587–596.[CrossRef][Medline]
van Dijk, E., Cougot, N., Meyer, S., Babajko, S., Wahle, E., Seraphin, B. (2002). Human Dcp 2, a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J 21, 6915–6924.[CrossRef][Medline]
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