Yeast contain a distinct RNA granule with composition similar to mammalian stress granules
An unresolved issue is whether the eIF4E-, eIF4G-, and Pab1p-containing granules observed in Saccharomyces cerevisiae
are analogous to mammalian stress. To determine this, we examined if they contained orthologues of additional mammalian proteins known to accumulate in stress granules using strains bearing a chromosomal C-terminal GFP fusion at the ORF of interest (Huh et al., 2003
). Besides accepted yeast orthologues of mammalian stress granule proteins, other proteins were examined based on BLAST homology to other mammalian stress granule proteins (e.g., HuR and G3BP), or protein/genetic interaction data, where interactions with eIF4F factors, Pub1, Pbp1, and Ngr1 were given particular consideration. Each protein that formed foci was also examined for its localization to P bodies when coexpressing an Edc3-mCh or Dcp2-RFP fusion protein (unpublished data).
These experiments revealed that during glucose deprivation the yeast orthologues of many proteins seen in mammalian stress granules accumulate in cytoplasmic foci, which can be distinct from P bodies (). These included the translation initiation factors eIF4GI, eIF4GII, eIF4E, and Pab1, Pub1, and Ngr1, which are the yeast orthologues of mammalian TIA-1 and TIA-R, and Pbp1, which is the orthologue of Ataxin-2, a component of mammalian stress granules required for their assembly (Nonhoff et al., 2007
; ). Several other proteins also accumulate in these foci (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807043/DC1
), including Hrp1 and Gbp2, which are nuclear-cytoplasmic shuttling proteins; Nrp1 and Ygr250c, predicted RNA binding proteins; and Eap1, an eIF4E binding protein (Cosentino et al., 2000
Figure 1. Candidate yeast stress granule proteins form P body–distinct cytoplasmic foci during glucose deprivation. Log-phase wild-type cells expressing chromosomal GFP-tagged proteins and Edc3-mCh (pRP1574) were glucose deprived and examined. No GFP foci, (more ...)
To determine if all these proteins were components of a single granule type, we examined their colocalization with a Pub1-mCh fusion protein. We observed that Pub1-mCh colocalized almost completely with GFP fusions of Pab1, eIF4GI, eIF4GII, eIF4E, Pbp1, Ngr1, Eap1, Hrp1, Ygr250c, Gbp2, and Nrp1 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807043/DC1
). This colocalization indicates that all these proteins are components of a single granule with a similar composition to mammalian stress granules. We refer to these granules as yeast stress granules and note that they are almost certainly the same RNP granules identified earlier as EGP bodies (Brengues and Parker, 2007
; Hoyle et al., 2007
In contrast to results with mammalian cells (Anderson and Kedersha, 2006
), yeast stress granules did not contain the Prt1 subunit of eIF3 (unpublished data) or the α-subunit of eIF2, the latter of which forms distinct rod-shaped foci during glucose deprivation (; Campbell et al., 2005
). This suggests that the multiple different types of mRNPs can accumulate to form stress granule–like RNPs under different conditions and in different organisms. Because stress granules are dynamic assemblies of mRNPs, this implies that the specific composition observed in the granule will depend on which step in a given mRNP transition process is rate limiting, which may change under differing cellular conditions (see Discussion). Additionally, mammalian P bodies often appear docked on the periphery of stress granules, whereas in yeast, when colocalization between P-body and stress granule components occurred, the overlap was more complete.
We also noted that the translation termination factors eRF1 and eRF3 accumulated in P bodies in a subset of cells, although the majority of these proteins remain cytoplasmic (Fig. S1). This is in agreement with the finding that eRF3 localizes in P bodies during stationary phase (Dori and Choder, 2007
), and suggests that translation termination may be coupled to assembly of P-body mRNPs.
Properties of yeast stress granules
Additional experiments revealed several properties of yeast stress granules. First, as in mammalian cells, the assembly of yeast stress granules is blocked by trapping mRNAs in polysomes with cycloheximide (), arguing that yeast stress granules require nontranslating mRNA for assembly, which is also supported by the fact that these granules contain mRNAs (Hoyle et al., 2007
). Second, unlike mammalian cells, we observed that several other stresses including oxidative stress (3 mM H2
for 15 min), hypo-osmotic stress (incubation in H2
O + dextrose), and hyper-osmotic stress (incubation in 1 M KCL + dextrose) had little or no effect on the induction of yeast stress granules, although all of these stresses increased P bodies to varying degrees (). This suggests that most stresses in yeast that decrease translation generally lead to the accumulation of mRNAs in P bodies.
Figure 2. Yeast stress granules are sensitive to cycloheximide, and stimulated by a constitutively active allele of the GCN2 kinase. (A) Log-phase yRP840 cells transformed with pRP1659 were glucose deprived and examined. Cycloheximide-treated cells (100 μg/ml) (more ...)
We also examined the effect upon stress granule formation in yeast of expressing a constitutively active allele of the Gcn2 kinase (Gcn2c; Ramirez et al., 1992
), which phosphorylates and decreases eIF2α function. The logic of this experiment was that in mammalian cells, stress granule assembly is often stimulated by phosphorylation of eIF2α at position Ser51 by stress-inducible kinases such as Gcn2 (Kedersha et al., 1999
). This phosphorylation leads to decreased translation initiation rates, causing accumulation of nontranslating mRNAs in stress granules. We observed that Gcn2c expression in mid-log cultures slightly increased P-body size and brightness over the vector control, and occasional stress granules were visible, though these almost always localized with P bodies (; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200807043/DC1
). More strikingly, in the presence of Gcn2c, both stress granules and P bodies were strongly enhanced during glucose deprivation (), with stress granules being brighter, larger, and more frequent in cells compared with those expressing an empty vector control. This suggests that, as in mammalian cells, accumulation of mRNAs stalled in translation initiation due to decreased eIF2 function leads to increased formation of stress granules.
Specific proteins affect yeast stress granule assembly
To determine how yeast stress granules assemble, we examined how the absence of specific proteins affected the assembly of either stress granules during glucose deprivation (monitored using Pab1-GFP or Pub1-mCh) or P bodies (monitored with Edc3-mCh). An initial set of null strains were chosen based on homology to factors reported to affect mammalian stress granule assembly (Kedersha et al., 1999
; Nonhoff et al., 2007
). We then quantified the effects of specific mutations on stress granules by blind scoring of the percentage of cells with stress granules, the average number of granules per cell, and the average size of stress granule foci (see Materials and methods, and Table S1). In these experiments, the absence of stress granules was not due to large changes in expression of the marker protein as verified by Western blot analysis or overall fluorescence levels (Fig. S3 [available at http://www.jcb.org/cgi/content/full/jcb.200807043/DC1
] and unpublished data). These experiments revealed the following important points.
First, we observed that pbp1Δ
strains showed a strong reduction in stress granule formation as judged by both the number of cells exhibiting Pab1 and Pub1 foci, and the average number of foci observed, although P bodies still formed at wild-type levels (; and see ). This is consistent with studies of the mammalian orthologue Ataxin-2, where siRNA depletion of the protein leads to reduction in stress granules, but has no effect on P-body formation (Nonhoff et al., 2007
Figure 3. Mutations that inhibit stress granule assembly do not significantly affect P-body assembly. Log-phase BY4741 wild-type and isogenic knockout strains were transformed with pRP1657, glucose deprived, and examined for altered stress granule/P-body assembly. (more ...)
Figure 6. Formation of Pub1-mCh foci predominantly mirrors null strain sensitivity trends exhibited by Pab1-GFP. Various yeast deletion strains and wild-type isogenic controls were transformed with pRP1661 or pRP1662, according to strain auxotrophies. Log-phase (more ...)
Second, we observed that pub1Δ
strains also displayed a strong decrease in the average number and percentage of cells with stress granules as judged by Pab1 localization, but with little effect on P bodies. Pub1 is the yeast homologue of TIA-1, which is thought to self-aggregate and promote stress granule assembly by virtue of a glutamine-rich prion domain (Kedersha et al., 1999
), which is conserved in Pub1 (Michelitsch and Weissman, 2000
). In contrast, strains lacking the yeast orthologue of TIA-R, Ngr1, did not show a defect in stress granule formation (), consistent with prior observations in mammalian cells (Gilks et al., 2004
). The similar role in stress granule assembly for Pbp1, Pub1, and their orthologues in mammalian cells strongly argues that yeast and mammalian stress granules are related structures.
Third, we observed that strains with decreased levels of eIF4G showed decreases in the amount of stress granules formed, again with no clear defects in P-body formation. Yeast has two genes for eIF4G. Strains lacking eIF4GII showed a strong decrease in stress granules as judged by both Pab1p and Pub1, although the degree of inhibition was not as severe as that of either pbp1Δ
strains (, and see ). In contrast, strains lacking eIF4GI showed a strong reduction in stress granules as judged by Pab1p (), but had no effect on Pub1 foci (see ). These results indicate that both eIF4G1 and eIF4GII can affect the assembly of stress granules and that the recruitment of Pub1p into stress granules is differentially affected by the different eIF4G paralogues. Interestingly, although P bodies increase with defects in certain initiation factor mutants (ts alleles of eIF4E, eIF3, and pab1Δ
; Teixeira et al., 2005
; Brengues and Parker 2007
), we did not observe an increase in P bodies with eIF4GIΔ
strains, which may reflect redundancy between these paralogues, or as-yet unappreciated roles for specific initiation factors in mRNP transitions between P bodies and stress granules.
These results identify Pub1, Pbp1, and eIF4G proteins as playing important roles in the assembly of yeast stress granules, while also critically demonstrating that yeast P bodies can form independently of stress granules.
Stress granule formation is not required for translation repression or mRNA stabilization
Stress granule formation occurs during stresses that lead to a reduction in translation initiation rates, and it has been proposed that the formation of the stress granules might play a role in such translation repression (Kedersha and Anderson, 2002
; Anderson and Kedersha, 2008
). In addition, many stresses globally stabilize mRNAs (Gowrishankar et al., 2006
; Hilgers et al., 2006
), and it has been inferred that such stabilization might be due to stress granule formation (Kedersha et al., 2005
; Stöhr et al., 2006
). Using the mutants that prevented the formation of stress granules, we examined whether stress granule formation affected translation repression and/or mRNA stabilization during glucose deprivation in yeast.
We observed that the pub1Δ, pbp1Δ, eIF4GIΔ, and eIF4GIIΔ strains, all of which are defective in stress granule formation, were able to repress translation similar to wild-type strains in response to glucose deprivation as judged by the incorporation of S35 into new proteins (; unpublished data). This observation indicates that stress granule formation is not required for global translation repression during glucose deprivation.
Figure 4. Stress granule assembly mutants are not deficient in their ability to both translationally repress and stabilize mRNA during stress. (A) Log-phase BY4741 wild-type and isogenic knockout strains were washed and incubated in media +/− glucose (more ...)
We also observed that the MFA2pG reporter mRNA, which is stabilized in response to glucose deprivation in yeast (Hilgers et al., 2006
), was also clearly stabilized in wild-type, pub1Δ
, and eIF4GIIΔ
strains (). This indicates that stress granule formation is not required for the major stabilization of mRNAs that occurs during glucose deprivation. Additionally, no differences in MFA2pG decay in the absence of stress were observed in any of the stress granule assembly mutants (). Thus, stress granule assembly is unlikely to function in protecting mRNAs from premature or aberrant decay.
Yeast stress granule formation is dependent on P-body assembly
An unresolved issue is the nature of interactions between stress granules and P bodies. During translation repression, mRNAs may be initially routed to stress granules, in which mRNAs are either retained in storage before reentry into translation, or are instead sent to P bodies for decay (Anderson and Kedersha 2006
). An alternative possibility is that mRNAs exiting translation first assemble into an mRNP that can accumulate in P bodies, followed either by degradation, retention for storage, or reentry into translation, which might lead to the formation of stress granules when steps in translation initiation are inhibited. As discussed above, the observation that P bodies can form in the absence of stress granules argues that both P bodies and stress granules form independently, or that P bodies are precursors to stress granules. To distinguish these two possibilities, we examined how defects in P-body assembly affected stress granule formation by taking advantage of mutant strains that inhibit P-body formation by limiting aggregation of mRNPs into larger P bodies (Coller and Parker, 2005
; Decker et al., 2007
). All images were quantified in a blind manner for the effects of each mutation on the percentage of cells with stress granules or P bodies, as well as average foci size and the average number of each foci per cell (see Materials and methods and Table S1).
To examine possible effects of P-body formation of stress granules, we first examined the effects of deletions of Edc3, which affects P-body aggregation, or the C-terminal tail of Lsm4, which has a prion-type domain that contributes to aggregation of P bodies (Decker et al., 2007
). In addition, we examined the effects of an edc3Δ lsm4Δc
double mutant where P bodies are dramatically reduced. An important result was that edc3
yeast, which are greatly reduced in P-body formation, were similarly strongly inhibited for formation of stress granules as judged by Pab1-GFP () or Pub1-mCh (see ). Occasional Pab1 or Pub1 foci were seen in a small fraction of cells, but they were fainter, smaller (Table S1), and at least in the case of Pab1, typically colocalized with what faint P bodies remained visible (). The edc3Δ
strains also showed reductions in stress granule formation, but similar to their partial effects on P-body formation, stress granules were reduced but could still form in edc3Δ
strains (). We interpreted these observations to suggest that the formation of stress granules was enhanced by P bodies.
Figure 5. Mutations that inhibit P-body formation also inhibit stress granule assembly. (A) Various yeast deletion strains and wild-type isogenic controls were transformed with pRP1657, pRP1658, or pRP1659, according to auxotrophies and genetic properties of the (more ...)
To extend this analysis, we wished to examine additional mutants with defects in P-body assembly or mutants lacking individual P-body protein components. Because edc3Δ lsm4Δc
strains can still form a few P bodies, we desired to create a strain with an even stronger defect in P-body assembly. We hypothesized that an edc3Δ pat1Δ
strain might be extremely defective in P-body formation because it would (1) lack Edc3; (2) lack Pat1, which can contribute to the formation of P bodies (Teixeira and Parker, 2007
); and (3) would be unable to use the Lsm4 C-terminal domain to assemble P bodies because the recruitment of the Lsm1-7p complex to P bodies is dependent on Pat1p (Teixeira and Parker, 2007
). Indeed, examination of an edc3Δ pat1Δ
strain showed an even stronger block to P-body formation than the edc3Δ lsm4Δc
strain, although a few cells could still form small and faint P bodies (). Moreover, the edc3Δ pat1Δ
showed an even stronger defect in stress granule formation (). This result identifies the edc3Δ pat1Δ
strain as the most defective strain for P-body formation, and provides additional evidence that P bodies are required for stress granule formation.
Further experiments revealed that strains lacking either Pat1 or Dhh1 proteins also showed defects in stress granule formation. In pat1Δ
strains, P-body formation was reduced and stress granules showed a corresponding reduction ( and ). Interestingly, in dhh1Δ
strains, stress granules were reduced compared with wild-type strains, yet P-body formation was normal (). This suggests that Dhh1 might have a specific role in stress granule assembly. Finally, examination of a dhh1Δ pat1Δ
strain, known to be deficient in both P-body formation and the ability to translationally repress (Coller and Parker, 2005
), revealed a strong block to stress granule formation ( and ). P bodies were significantly decreased in number, though not as severely as in edc3
Δ strains, arguing that in this case, a combination of impaired P-body assembly, fewer mRNAs exiting translation, and/or an inability to transition from P bodies to stress granules may account for this defect.
These results indicate that formation of stress granules in yeast is enhanced by existing P bodies. Moreover, in all these cases, the reduction or absence of stress granules is not due to large changes in Pab1p-GFP expression, as verified by Western blot (Fig. S3). One simple interpretation of these observations is that mRNAs move from P bodies to stress granules during transitions between different mRNP complexes, and that the formation of stress granules is facilitated by a preexisting pool of untranslating mRNPs in P bodies (see Discussion).
A prediction of stress granules forming from mRNAs in P bodies is that strains with enlarged P bodies should show enhanced assembly of stress granules. To test this prediction, we examined stress granule formation in strains defective in mRNA decapping (dcp1Δ
) and 5′ to 3′ degradation of mRNAs (xrn1Δ
), where P bodies are increased (Sheth and Parker, 2003
; Teixeira and Parker, 2007
). Strikingly, we observed that during glucose deprivation, stress granules were larger and more numerous in xrn1Δ
strains than in wild-type cells (). Interestingly, in dcp1Δ
strains, Pab1 often formed donut-like structures with Edc3 foci visible at the center (). These results are consistent with the mRNAs in stress granules predominantly being derived from P bodies. We also observed that both stress granules (as judged by Pab1 and Pub1) and P bodies (as judged by Edc3) were more diffuse in xrn1Δ
strains than in wild-type cells, suggesting some alteration in their underlying morphology (). Interestingly, both dcp1Δ
strains showed significantly more Pub1 foci than Pab1 foci in the absence of stress (, , and Table S1), suggesting that the kinetics of mRNA binding and dissociation differs between Pab1 and Pub1. One explanation for this may be that under nonstress conditions, Pub1 is removed from mRNAs in P bodies as the mRNA is degraded.
Temporal analysis indicates stress granules first form in conjunction with P bodies
The dependence of stress granule assembly on P-body formation suggests that stress granules may form by mRNPs in P bodies exchanging proteins to form an mRNP poised to reenter translation. This model makes two predictions. First, it predicts that stress granules would form after P bodies during stress responses. Second, it predicts that the components of stress granules would first associate with mRNPs in P bodies, followed by maturation of such a complex into a stress granule, as the translation repression/decapping complex was exchanged for translation factors. To test these predictions, we followed the formation of yeast stress granules and P bodies during glucose deprivation by time-lapse microscopy using Pab1-GFP as a marker of stress granules and Dcp2-mCh or Edc3-mCh as a marker of P bodies.
We observed that from 0–7 min after the onset of glucose deprivation stress, P bodies were strongly induced whereas stress granules, as judged by Pab1-GFP, were predominantly absent. However, wherever faint foci were present, they were colocalized with P bodies (; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200807043/DC1
). As time progressed, Pab1-GFP foci became brighter within P bodies and then started to form additional cytoplasmic foci which were separate from bright preexisting P bodies. Interestingly, a very faint signal of Dcp2-mCh or Edc3-mCh was often colocalized with these newly “P body–distinct” stress granule foci (; unpublished data). Additionally, P bodies that become enriched with Pab1 often showed a partially decreased signal for Dcp2-mCh or Edc3-mCh over time, whereas the Pab1 signal maintains or intensifies, suggesting some type of maturation process is indeed occurring. These observations indicate that yeast stress granules form after P bodies, primarily in conjunction with preexisting P bodies.
Figure 7. Yeast temporal analysis reveals accumulation of Pab1 in P bodies before formation of P body–distinct stress granules. (A) Log-phase yRP840 strain, transformed with pRP1660, was glucose deprived for 5 min and followed over time. For technical reasons, (more ...)
Induction of mammalian P bodies and stress granules share kinetic properties with yeast
The above results demonstrate that stress granules in yeast form after P bodies during stress and require existing P bodies for efficient accumulation. To examine the relationship between stress granule formation and P bodies in mammalian cells, we examined a time course of stress granule and P-body formation in HeLa cells after arsenite stress using Pabp and Rck (Dhh1 homologue) as markers of stress granules and P bodies, respectively. Similar to yeast cells, we observed that after arsenite stress in HeLa cells, P bodies first increase in number, size, and intensity within 10 min, with no initial change in Pabp distribution (). However, in contrast to the strong colocalization of Pab1-GFP and P-body foci when stress granules are first forming in yeast, stress granules in mammalian cells primarily first appear as small foci distinct from P bodies, although occasional colocalization of Pabp and Rck was seen. At later time points, “classical” morphology stress granules appear that are usually docked to P bodies (, “45 min”). These results suggest that stress granules in mammalian cells may form independently of P bodies, or that the biochemical activity of P bodies that enhances stress granule formation may be present in multiple small P bodies throughout the cytosol in mammalian cells (see Discussion).
Figure 8. Temporal analysis of arsenite-stressed HeLa cells reveals P bodies increase in size and number before formation of stress granules. (A) HeLa cells were fixed at several time points after arsenite stress, from 0 (no arsenite added) to 60 min. Endogenous (more ...)
We also observed that Rck, the mammalian orthologue of yeast Dhh1, accumulated in P bodies for the first 45 min of arsenite stress in HeLa cells, but then at 1 h began to accumulate in large stress granules also (). This accumulation of Rck in stress granules at late time points during stress was also recently observed in another study (Wilczynska et al., 2005
; Mollet et al., 2008
). Although speculative, one possibility is that Rck may associate with mRNAs in P bodies and then play a role in those mRNAs transitioning into stress granules, perhaps as an intermediate in an mRNP remodeling process. Together, these data suggest that like yeast, at least some mammalian mRNAs may transition through P bodies before accumulating in stress granules.