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RESEARCH ARTICLE

mRNA localization to P-bodies in yeast is bi-phasic with manymRNAs captured in a late Bfr1p-dependent wave

Clare E. Simpson1, Jennifer Lui2, Christopher J. Kershaw2, Paul F. G. Sims3 and Mark P. Ashe2,*

ABSTRACT

The relocalization of translationally repressed mRNAs to mRNA

processing bodies (P-bodies) is a key consequence of cellular

stress across many systems. P-bodies harbor mRNA degradation

components and are implicated in mRNA decay, but the relative

timing and control of mRNA relocalization to P-bodies is poorly

understood. We used the MS2–GFP system to follow the movement

of specific endogenous mRNAs in live Saccharomyces cerevisiae

cells after nutritional stress. It appears that the relocalization of

mRNA to P-bodies after stress is bi-phasic: some mRNAs are

present early, whereas others are recruited much later concomitant

with recruitment of translation initiation factors, such as eIF4E. We

also find that Bfr1p is a late-phase-localizing P-body protein that is

important for the delayed entry of certain mRNAS to P-bodies.

Therefore, for the mRNAs tested, relocalization to P-bodies varies

both in terms of the kinetics and factor requirements. This work

highlights a potential new regulatory juncture in gene expression

that would facilitate the overall rationalization of protein content

required for adaptation to stress.

KEY WORDS: P-bodies, Stress granules, Glucose regulation,

mRNA localization, Yeast

INTRODUCTIONStringent regulation of mRNA translation and degradation is

fundamental in allowing eukaryotic cells to control their diverse

protein content. These mechanisms become especially important

following stress; cells must decrease their energy consumption

while accumulating proteins that are required for adaptation

(Pichon et al., 2012; Simpson and Ashe, 2012). mRNA

processing bodies (P-bodies) are induced under such stress

conditions and represent sites where components of the 59 to 39

mRNA decay pathway are concentrated (Buchan et al., 2010;

Kedersha et al., 2005). As this pathway serves as a major route for

bulk mRNA degradation, P-bodies are considered sites where

mRNA can be degraded, particularly after stress (Balagopal et al.,

2012; Balagopal and Parker, 2009; Buchan et al., 2010). P-bodies

also have key functions during embryonic patterning (Weil et al.,

2012), viral infection (Beckham and Parker, 2008), micro-RNA-mediated decay (Pillai et al., 2005), and nonsense- or AU-rich-element-mediated decay (Durand et al., 2007; Fenger-Grøn et al.,

2005).

In addition to P-bodies, several other classes of mRNA-containing granule have been identified. These include stress

granules, which are found in many cell types, and neuronalgranules and P-granules, which have been found in neurons andembryonic cells, respectively (Thomas et al., 2011; Updike and

Strome, 2010). These granules occur in a variety of conditionsand they contain many overlapping components, such as mRNAs,mRNA-binding proteins and proteins associated with translation

inhibition (Kedersha and Anderson, 2009; Parker and Sheth,2007). Previously, we and others have characterized granules thatharbour mRNA and translation initiation factors that accumulate

as a response to stress in Saccharomyces cerevisiae (Buchanet al., 2008; Hoyle et al., 2007). We studied the relocalization ofthe eukaryotic translation initiation factors, eIF4E, eIF4G andPab1p both to P-bodies and to stress-induced granules (EGP-

bodies), which contain these select mRNA-associated translationinitiation factors but, crucially, lack the components of themRNA decay machinery that are associated with P-bodies (Hoyle

et al., 2007). Other stress granules have been identified that formin response to very severe stress in yeast, and these granules aremuch more akin to mammalian stress granules, at least in terms of

composition (Grousl et al., 2009; Grousl et al., 2013; Kato et al.,2011). Therefore, the relationship between the variouscomponents of P-bodies and other RNA granules is complex

and is thought to be dynamic, meaning proteins and mRNAs areable to move between granule subsets (Buchan et al., 2010;Kedersha et al., 2005). For instance, studies have shown thatmRNAs have the ability to re-enter the translational pool

following relief from stress (Brengues et al., 2005) and that thisability might be specific to certain mRNAs, occurring over afinite period (Arribere et al., 2011).

mRNA-binding proteins are well established to play crucialroles in determining mRNA fate and are associated with RNA

granule formation. For instance, Edc3p, Pat1p and the Lsm1–7pbinding complex function in P-body regulation, whereas theRNA-binding protein TIA-1 (Pub1p in yeast) is necessary for

stress granule formation (Decker et al., 2007; Duttagupta et al.,2005; Kedersha et al., 1999; Reijns et al., 2008). Furthermore, thetristetrapolin protein in mammalian cells is thought to regulateassociation of the AU-rich cytokine mRNAs with P-bodies

(Franks and Lykke-Andersen, 2007). The association of a varietyof RNA-binding proteins with P-bodies not only suggests thatmany mRNAs are localized here, but also highlights the

possibility that the localization of mRNAs might bedifferentially regulated. Although the targeting of mRNA to P-bodies has been studied for specific mRNAs (Arribere et al.,

2011; Hoyle et al., 2007; Lavut and Raveh, 2012; Sheth and

1Department of Biochemistry, Downing Site, The University of Cambridge,Cambridge CB2 1QW, UK. 2The Faculty of Life Sciences, The Michael SmithBuilding, The University of Manchester, Oxford Road, Manchester M13 9PT, UK.3Faculty of Life Sciences, Manchester Institute of Biotechnology (MIB), TheUniversity of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

*Author for correspondence (mark.p.ashe@manchester.ac.uk)

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distributionand reproduction in any medium provided that the original work is properly attributed.

Received 25 July 2013; Accepted 8 December 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1254–1262 doi:10.1242/jcs.139055

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Parker, 2006), key questions regarding the timing and specificityof mRNA recruitment remain unanswered.

In this study, we have used the m-TAG system to investigatemRNA relocalization after stress in live yeast cells.Characterization of numerous mRNAs in this manner revealedthat there are at least two distinct phases of mRNA localization to

P-bodies. Firstly, there are those mRNAs that are present in RNAgranules early after glucose starvation. Later, there is a secondphase of mRNA recruitment to P-bodies after more extended

periods of stress, which is reliant upon the earlier formation of P-bodies. A screen for Xrn1p–Dcp2p interacting proteins identifieda P-body protein, Bfr1p, that, similar to the late mRNAs, localizes

to P-bodies in a delayed manner. Furthermore, deletion of theBFR1 gene prevented the late-phase entry of specific mRNAs toP-bodies suggesting that this mRNA-binding protein plays an

important role in the regulation of mRNA fate following glucosedepletion.

RESULTSThere are two mRNA localization profiles relative to P-bodiesfollowing glucose starvationIn order to inspect and follow the localization of specific mRNAs

in live S. cerevisiae cells, we used the m-TAG system (Haimet al., 2007). A detailed explanation of this technique has beenprovided elsewhere (Haim-Vilmovsky and Gerst, 2009);

however, in short, the genomic copy of an mRNA sequence istagged within its 39UTR with MS2 stem loops. This allows thevisualization of the mRNA via coexpression of the MS2 coat

protein fused to three green fluorescent proteins (CP–GFP3). Suchsystems have been used extensively to examine the localization ofmRNAs in a wide range of biological systems (Haim et al., 2007;Hamada et al., 2003; Sheth and Parker, 2006). Key advantages of

this yeast system are that the control elements associated withmRNA transcription and processing [promoters, UTRs, poly(A)sites and terminators] remain intact, as the MS2-binding sites are

inserted directly and precisely into the 39UTR of the endogenousgene at its chromosomal locus (Haim et al., 2007). A potentiallimitation of the approach is that the insertion of the MS2 stem

loops could alter aspects of the behavior of an mRNA; however, anumber of mRNAs have been functionally evaluated after MS2insertion and found to be unaffected (Haim et al., 2007).Consequently, using this system, the localization of an mRNA

can be evaluated in live cells, allowing responses to changingexternal cues to be assessed.

At the outset, we tagged numerous mRNA sequences with MS2

stem loops; these mRNAs were selected because they are highlyabundant and the protein products are associated with a variety offunctions (supplementary material Table S1). It might be

predicted that the addition of the MS2 stem loops would increasethe stability of the mRNA; however, most of the resulting strainsexhibited little difference in the expression level of the MS2-

tagged mRNAs relative to the level of untagged mRNA in wild-type strains as judged by quantitative reverse transcription (qRT)-PCR. Levels of the PGK1 and RPS16A mRNAs actuallydecreased with the added MS2 sequences (supplementary

material Fig. S1). It is currently unclear how this decrease inmRNA level occurs. Nevertheless, levels of the MS2-taggedmRNAs being tested are either wild type or lower than this. The

MS2-tagged mRNA strains also contained the markers Dcp2p–cyan fluorescent protein (CFP) and Cdc33p–red fluorescentprotein (RFP). Dcp2p is the catalytic subunit of the decapping

enzyme and serves as a marker for P-bodies, whereas Cdc33p is

the eIF4E translation initiation factor that binds the mRNA capand enters both P-bodies and EGP-bodies (stress granules). In

these tagged strains, therefore, we define EGP-bodies asharboring eIF4E but not the P-body marker Dcp2p.

From the outset, it was evident that mRNAs fell into twocategories of mRNA localization. The first class of mRNA

observed using the m-TAG system is characterized by mRNAsthat colocalized with the P-body marker Dcp2–CFP early after P-bodies have formed, that is after 10 minutes of glucose starvation

(Fig. 1). These mRNAs also exhibited no real increase in thelevel of P-body association from 10 to 50 minutes post glucosestarvation. Furthermore, there was little evidence that these

mRNAs accumulated in granules that harbor eIF4E but lackDcp2p, i.e. EGP-bodies/stress granules. Examination of thelocalization of these mRNAs under non-stress conditions

revealed that here too they were present in mRNA granules(supplementary material Fig. S2). Under these non-stressconditions, neither the mRNA decay components nor thetranslation initiation factors exhibit any granular localization

(Hoyle et al., 2007 and data not shown). A detailed characterizationand functional analysis of the mRNA granules that are present inexponentially growing cells will be published elsewhere. In this

current study, we have focused on the localization of mRNAs toP-bodies and stress granules, and this class of mRNA enters P-bodies early after their formation. Two of these mRNAs,

RPS16A and RPS23B, encode ribosomal proteins. A rapidtargeting of mRNAs encoding ribosomal proteins to P-bodies,most likely for degradation, agrees with previous research

showing that such mRNAs rapidly diminish in polysomes afterglucose starvation (Arribere et al., 2011). Another mRNA inthis class is PGK1, which encodes the glycolytic enzymephosphoglycerate kinase. Previously, using a plasmid-U1A-

based mRNA-localization strategy, we have shown that thePGK1 mRNA partially colocalized with eIF4E in granules(Hoyle et al., 2007). Here, using the m-TAG system in strains

where both eIF4E and Dcp2p can be simultaneously visualized,we determined that the PGK1-mRNA-containing granules are P-bodies that also contained eIF4E (Fig. 1). Overall, this first class

of mRNA is present early in P-bodies and does not localize toEGP-bodies/stress granules.

We also identified a second class of mRNA that displayeddifferent localization kinetics. These mRNAs were unlocalized

both under non-stress conditions (supplementary material Fig.S2) and after 10 minutes of glucose depletion; a time when P-bodies had already formed, as judged by the localization of

Dcp2p–CFP (Fig. 2). Therefore, at this early stage the majority ofP-bodies lacked the tagged mRNA. However, after 50 minutes ofglucose starvation, localization of these mRNAs to P-bodies

occurred (Fig. 2). As for the early class of mRNAs, there is littleevidence that these mRNAs accumulated in granules that harboreIF4E but lack Dcp2p, i.e. EGP-bodies/stress granules. Thus, it

appears that this second class of mRNA localizes to P-bodies overan extended time after stress, and does not localize to EGP-bodies(stress granules).

A protracted entry of mRNA into P-bodies is consistent with a

current model for translational repression following glucosestarvation (Castelli et al., 2011). In this model, a loss of the eIF4ARNA helicase from the mRNA would inhibit translation

initiation. However, over a short period after the stress, theeIF4A loss would cause the 48S preinitiation complex toaccumulate. Over a more prolonged period, this stalled 48S

complex would break up, allowing the slow release of mRNA

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Fig. 1. Early-phase mRNAs are present in P-bodies early after glucose depletion. Fluorescence microscopy images of yeast cells at two different timepoints after glucose depletion. The RPS16A, RPS23B and PGK1 mRNAs are followed using the m-TAG system (MS2–GFP), mRNA decay componentsare followed using CFP-tagged Dcp2p and components of the closed loop complex are followed using RFP-tagged eIF4E across the same cells. Thecolored inset overlay images after 50 minutes of glucose depletion depict examples where the mRNAs colocalize with P-bodies (yellow triangles) but notwith EGP-bodies (white diamonds). Graphs to the right represent the percentage of P-bodies or EGP-bodies that harbor each mRNA after 10 minutes (whitebars) and 50 minutes (gray bars) of glucose depletion. Scale bars: 5 mm.

Fig. 2. Late-phase mRNAs enter P-bodies after an extended period of glucose starvation. As Fig. 1, following SPG4, SUE1, VNX1, TDP1 and RRP43

mRNA relative to mRNA decay and closed loop complex components. The colored inset overlay images after 50 minutes of glucose depletion depict exampleswhere the mRNAs colocalize with P-bodies (yellow triangles) but not with EGP-bodies (white diamonds). Graphs to the right show that the percentage of P-bodies harboring each mRNA increases from 10 minutes (white bars) to 50 minutes (gray bars) of glucose depletion, whereas minimal colocalization with EGP-bodies was observed. Scale bars: 5 mm.

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associated with components of the closed loop complex. This couldexplain why eIF4E only localized to P-bodies at 50 minutes, but

not 10 minutes, post glucose stress (Fig. 2) (Hoyle et al., 2007). Amore detailed quantification of the association of the MS2-taggedmRNAs and eIF4E with P-bodies suggested that for all eight MS2-tagged mRNAs evaluated in this study, only a small percentage of

the tagged mRNA was present in P-bodies that also contain eIF4E(supplementary material Fig. S3). Although it is still possible thatmRNAs enter P-bodies as part of the closed loop complex, this

quantification also highlighted an alternative possibility: that theclosed loop complex breaks down prior to, or during, themovement of these mRNAs to P-bodies.

In order to compare the timing of eIF4E and late-phase mRNAentry to P-bodies, a more comprehensive time course wasperformed using the TDP1 mRNA. Here, the earliest time point at

which localization to P-bodies is observable, for either eIF4E orthe TDP1 mRNA, was 30 minutes post glucose starvation(Fig. 3). This apparent coincidence in the timing of movementfor closed loop complex translation initiation factors and a late-

phase mRNA supports a model where at least some of the mRNAmoves to P-bodies while still associated with the closed loopcomplex, following a protracted association with the translational

machinery after glucose starvation. An obvious question fromthis comparison of late mRNA and eIF4E entry to P-bodies iswhy does eIF4E not move into P-bodies with the early mRNAs.

A possible explanation is that the translationally repressedmessenger ribonucleoprotein particle (mRNP) complex for theearly mRNAs differs from that of the late mRNAs, and that, as a

result, the mechanism of transfer to P-bodies differs.

Localization of late-phase mRNAs requires P-bodiesIn order to explore the mechanistic requirements for these two

phases in mRNA recruitment to P-bodies, we made use of thelsm4DC edc3D mutant, which fails to form P-bodies. Lsm4p andEdc3p proteins contain specific domains that are essential for P-

body formation, presumably as a consequence of their potentialfor aggregation (Decker et al., 2007). Therefore, strains bearingthe various MS2-tagged mRNAs were individually backcrossed

to lsm4DC edc3D mutant strains. As expected, for all of theresulting lsm4DC edc3D mutant strains, P-bodies did not form

after either 10 or 50 minutes of glucose depletion (Fig. 4). Late-phase mRNAs, such as TDP1 and VNX1, were not localized at

either early or late time points post glucose starvation in themutant background. This result suggests that localization of theselate-phase mRNAs is dependent on the formation of P-bodies(Fig. 4). In contrast, RPS16A mRNA was observed in granules in

the complete absence of P-body formation. This observation mostlikely relates to the localization of these mRNAs to granules inunstressed cells, where P-bodies are not observed. These results

do suggest that the distinction between the early- and late-phasemRNAs lies not only in the timing of the localization but also inthe precise molecular mechanisms of the mRNA localization for

each class of mRNA.

Bfr1p is a late entry P-body proteinIn order to screen for factors co-purifying with the mRNA decayfactors Xrn1p and Dcp2p, we used tandem affinity purification(TAP) chromatography to pull down Dcp2p–TAP and Xrn1p–TAP from appropriate TAP-tagged strains. Many interacting

proteins were identified that have connections with RNA, or areassociated with P-bodies; these will be presented in greater detailelsewhere. A particularly prominent protein that was identified by

mass spectrometry in these pull downs was Bfr1p (Table 1).Bfr1p is an mRNA-binding protein that was initially identified asa high-copy suppressor of the lactone antibiotic brefeldin-A

(Jackson and Kepes, 1994). More recent studies have shown thatBfr1p interacts with an mRNA-binding complex containingRNA-binding proteins, such as Scp160p (Lang et al., 2001;

Scheuner et al., 2001; Sezen et al., 2009). Both Scp160p andBfr1p colocalize with the yeast endoplasmic reticulum (ER) in acharacteristic pattern around the nucleus (cortical ER) and justunder the cell membrane (peripheral ER) (Mitchell et al., 2013;

Sezen et al., 2009). The Bfr1p-containing complex has also beenshown to localize to polysomes in exponentially growing cells,suggesting that it is actively involved in regulating translation

(Sezen et al., 2009). On this basis, the potential interactionbetween Bfr1p and mRNA decay factors present in P-bodies wasfurther investigated.

TAP-affinity purifications on extracts prepared from strainsharboring Bfr1p–TAP revealed that Myc-tagged Xrn1p, and a

Fig. 3. Time course of localization of a late-phase mRNA to P-bodies relative to eIF4E.Fluorescence microscopy images of yeast cellsover a time course after glucose depletion. TheTDP1mRNA is followed using the m-TAG systemvia MS2–GFP (middle row), mRNA decaycomponents are followed using CFP-taggedDcp2p (top row) and components of the closedloop complex are followed using RFP-taggedeIF4E (bottom row). A white triangle highlightsthe time point where eIF4E and the mRNA arefirst observed colocalizing with the P-bodymarker. Scale bar: 5 mm.

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small amount of Myc-tagged Dcp2p, could be immunoprecipitatedwith Bfr1p (Fig. 5A). The eEF1A translation elongation factor(Tef1p) serves as a specificity control, as this protein is one of

the most abundant in the cell and is often a contaminant ofimmunoprecipitations (Krogan et al., 2006). Therefore, the absenceof this factor demonstrates the specificity of the interactions

between Bfr1p and the mRNA decay factors. The reciprocalpurification of either Xrn1p–TAP or Dcp2p–TAP also resulted inco-purification of Myc-tagged Bfr1p (Fig. 5B). Given that bothBfr1p and Xrn1p–Dcp2p have previously been described as being

associated with mRNA, or have been implicated in mRNAdegradation, the RNA dependence of the interaction wasassessed by treatment with RNase I. This treatment results in a

reduction in the level of co-immunoprecipitation suggesting thatmost of the interaction occurs via RNA (Fig. 5B).

As Bfr1p has been described as being associated with mRNAs

in polysomes, most likely at the ER (Mitchell et al., 2013; Sezenet al., 2009) and, here, we show that it interacts via mRNA withthe mRNA decay machinery, we considered the possibility that

this protein could act as a mediator involved in the transition frommRNA translation to mRNA degradation. Such a mediator mightbe expected to accumulate at the site of mRNA degradation. In

order to directly assess this, Bfr1p was GFP-tagged using agenomic tagging strategy (Janke et al., 2004). In exponentially

growing cells, GFP-tagged Bfr1p was observed both at theperiplasm (around the nucleus) and at the cell periphery(Fig. 5C), consistent with previous studies that demonstrate anER localization (Huh et al., 2003; Mitchell et al., 2013). A more

diffuse fluorescence was also observed throughout the cellularcytoplasm (Fig. 5C). After 10 minutes of glucose starvation, thepattern of Bfr1p localization became more diffuse, whereas after

50 minutes of glucose starvation, Bfr1p was observed to start toaccumulate in P-bodies (Fig. 5C). Intriguingly, this delayedrelocalization corresponded with that observed for the late-phase

mRNAs and the closed loop translation initiation factors,highlighting a possible role for Bfr1p in the transition of thelate-phase mRNAs from the translated pool to P-bodies.

Bfr1p is necessary for late-phase targeting of mRNAs to P-bodiesIn order to directly evaluate the role of Bfr1p in the transition ofmRNAs to P-bodies, the localization of specific mRNAs was

assessed in bfr1D mutant strains. In all cases, following glucosestarvation, P-body formation was still observed at early timepoints (Fig. 6A,B). The RPS16A mRNA, which localizes to P-

bodies almost immediately after their formation, was stillobserved in P-bodies (Fig. 6B,C). In contrast, two differentlate-phase mRNAs, VNX1 and TDP1, had not entered P-bodies

after 50 minutes of glucose starvation (Fig. 6B,C), or even aftermore protracted starvation periods (data not shown). Therefore,the Bfr1p mRNA-binding protein is essential for the targeting of

these late-phase mRNAs to P-bodies. This difference betweenlate and early mRNAs, in terms of their factor requirements,further exemplifies the mechanistic distinction between these twoclasses of mRNA.

DISCUSSIONCells change and adapt their proteome to cope with alterations

that occur in their surroundings. This is true for both the simpleunicellular organisms, like yeast, and cells from more complexmulticellular organisms (Simpson and Ashe, 2012; Spriggs et al.,

2010; Toone and Jones, 1998; Welch, 1987). For instance, tosurvive stresses, such as nutrient depletion, cells must reducetheir energy consumption, while rationalizing their proteincontent to adapt, in the shortest possible time, to the changing

conditions. In S. cerevisiae, as a response to glucose depletion,energy consumption is minimized by a rapid downregulation of avariety of energy-consuming processes, including protein

synthesis (Ashe et al., 2000), actin polymerization (Uesonoet al., 2004), tRNA nucleocytoplasmic export (Whitney et al.,2007) and endosomal trafficking (Aoh et al., 2011). In particular,

the rapid inhibition of translation initiation and subsequentappearance of P-bodies, combined with transcriptionalreprogramming has been viewed as a means by which cells can

Table 1. Summary of Bfr1p identification via mass spectrometry in affinity purifications of Dcp2p and Xrn1p

Bait PLGS score Number of peptides Coverage of Bfr1p (%)

Xrn1–TAP 564 21 37Dcp2–TAP 3251 54 50

Immunoprecipitations were performed on extracts from cells starved of glucose for 50 minutes and analyzed by mass spectrometry on a SYNAPTTM HDMSTM

system (Waters). The PGLS score is calculated by the Protein Lynux Global Server and is a statistical measure of the accuracy of assignation where higherscores imply greater confidence of protein identification (Xu et al., 2008).

Fig. 4. Late-phase mRNA localization relies upon P-body formation.Fluorescence images of yeast cells after 50 minutes of glucose depletion.lsm4DC edc3D mutant strains were generated that carry the MS2-taggedmRNAs labeled on the left. This mutant is deficient in P-body formation, asshown by the lack of localization for Dcp2p–CFP. The RPS16A mRNAprovides an example where early-phase mRNAs still aggregate, whereaslocalization is not observed for two late-phase mRNAs, VNX1 and TDP1.Scale bars: 5 mm.

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rapidly alter their gene expression profile (Arribere et al., 2011;

Lui et al., 2010). In this study, we provide further evidence for therationalization of mRNAs after stress by showing that there are atleast two phases in the mobilization of mRNA to P-bodies. Weshow that these two phases have differing requirements, both in

terms of P-body formation and the mRNA-binding protein Bfr1p,

which highlights the intriguing possibility that the phases couldbe independently controlled.

Following glucose starvation, translation initiation is rapidlyinhibited and, as a consequence, P-bodies form almost

Fig. 5. Bfr1p interacts with Dcp2p and Xrn1p via RNA and enters P-bodies. (A) Western blots (IB) from TAP (TAP-IP) on strains bearing TAP-tagged Bfr1p,as well as Myc-tagged Xrn1p or Dcp2p. (B) The reciprocal affinity purification of either Xrn1p–TAP or Dcp2p–TAP in strains containing Bfr1p–Myc. In bothA and B, TAP-tagged proteins were detected with a protein A peroxidase conjugate (PAP), Myc-tagged proteins were detected with an antibody against Myc,the presence of Tef1p was detected using an antibody against Tef1p. In both sets of experiments, an untagged wild-type strain was used as a negativecontrol on the same gel. (C) Localization of Bfr1p in exponentially growing cells (+ glucose) or after 10 or 50 minutes of glucose starvation (2 glucose). Scalebar: 5 mm.

Fig. 6. Late-phase mRNA localization to P-bodies requires Bfr1p. Images of bfr1D mutant strains containing Dcp2p–CFP and MS2-tagged RPS16A, VNX1or TDP1. The mRNA and Dcp2p localization is shown after (A) no starvation, (B) 10 minutes of glucose starvation, (C) 50 minutes of glucose starvation. Scalebars: 5 mm.

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immediately. Typically, the first class of mRNA that we haveidentified colocalized with P-body components instantly after P-

body formation. In contrast, a second class of mRNA localized toP-bodies gradually over an extended period of glucose starvation.More detailed kinetics reveal that the localization of mRNAcoincides with the timing of eIF4E, eIF4G and Pab1p

relocalization, which takes longer than 30 minutes (Hoyleet al., 2007). We have previously shown that glucose starvationleads to a rapid loss of the RNA helicase eIF4A from the

translation machinery and the accumulation of a 48S intermediatecomplex lacking eIF4A (Castelli et al., 2011). This complexpersists for over 30 minutes after glucose starvation, gradually

decaying such that the mRNA, and the translation factors closelyassociated with it, might only be available to localize to P-bodiesat later time points. Combined with the data presented here, this

leads to a model where the late-phase mRNAs localize to P-bodies gradually because of their prolonged association with thetranslation machinery. This could represent a mechanism bywhich the late-phase mRNAs remain translationally primed, such

that translation is inhibited without the mRNA being rapidlytargeted for decay. This would allow translational resumptionshould conditions become more favorable. Longer periods of

stress would allow the dissociation of the mRNA closed loop

complex, either in the P-body or during relocalization, such thatthe mRNA could be degraded (Fig. 7).

A precedent exists in terms of altered localization to P-bodiesover time for the catalytic subunits of the cAMP-dependentprotein kinase (PKA), Tpk2p and Tpk3p. Tpk2p localizes to P-bodies directly after glucose starvation with levels increasing

over time, whereas Tpk3p localization only occurs at later timepoints, concurrent with the second phase of mRNA localization(Tudisca et al., 2012). Intriguingly, PKA activity has also been

linked to P-body formation, in that the active PKA in glucose-replete cells supresses P-body formation via phosphorylation ofPat1p and, after glucose starvation, dephosphorylation of Pat1p

coincides with P-body formation (Ramachandran et al., 2011).Therefore, one possibility is that the regulated localization of thePKA catalytic subunits could play a role in the control of mRNA

mobilization to P-bodiesThe distinct nature of the two phases of mRNA localization to

P-bodies is further reflected by two additional findings. Firstly,mutations that prevent P-body formation do not impede the

localization of the early-phase mRNAs, whereas these mutationsdo prohibit the localization of the late-phase mRNAs. Secondly,deletion of the BFR1 gene prevents late-phase mRNA

localization without affecting the early-phase mRNAs, but inthis case P-bodies form normally. Bfr1p has previously beensuggested to play a role in the inhibition of mRNA translation via

Scp160p and its interaction with the eIF4E-binding protein (4E-BP) Eap1p (Lang et al., 2001; Sezen et al., 2009). Taken togetherwith the data in our study, this provides evidence that Bfr1p

might function as part of an intermediary mRNP complex thatdirects late-phase mRNAs from the translation machinery to themRNA decay system (Fig. 6). Studies aimed at defining themRNAs bound by a range of RNA-binding proteins suggest that

Bfr1p and Scp160p interact with in excess of 1000 mRNAs(Hogan et al., 2008). Hence, it is possible that although Bfr1p isrequired for late-phase entry to P-bodies, it does not provide the

specificity with which mRNAs are selected for this fate.However, the exact nature and composition of the late andearly mRNPs that enter P-bodies is unknown and will provide a

focus for further studies.Glucose starvation in yeast has also been shown to cause the

appearance of granules that harbor translation initiation factors andRNA-binding proteins but lack the mRNA decay machinery

(Buchan et al., 2008; Hoyle et al., 2007). These have been termedboth EGP-bodies or stress granules. Other severe stress conditionshave identified granules in yeast that are more similar to

mammalian stress granules, as they harbor eIF3 and the 40Sribosomal subunit (Grousl et al., 2009; Grousl et al., 2013; Katoet al., 2011). Of the mRNAs tested in this study, neither early- nor

late-phase mRNAs appear to localize to EGP-bodies followingglucose starvation, instead these mRNAs are almost exclusivelylocalized to P-bodies. Hence, even where the mRNA granule

colocalizes with a translation initiation factor, such as eIF4E, thisgranule will also contain components of the mRNA decaymachinery, such as Dcp2p. Therefore, we rarely observe themRNA in granules that do not contain the P-body markers after

glucose starvation. There are several possible explanations forthese observations: mRNAs might not enter EGP-bodies at all, theymight enter for a very transient period, or a very specific subset of

mRNAs might be present there. EGP-bodies contain thosetranslation initiation factors that are known to interact withmRNA, as well as at least three well-characterized RNA-binding

proteins (Buchan et al., 2011). Therefore it seems likely that

Fig. 7. A model depicting the two phases of mRNA relocalization to P-bodies. Phase I. Following cellular stress, such as glucose starvation, early-phase mRNAs relocalize to P-bodies with the mRNA decay machinery.Here, the mRNAs are either degraded or held in a translationally repressedstate. Phase II. More prolonged glucose starvation leads to a release oflate-phase mRNAs that have been associated with the translation initiationmachinery in a repressed state. These mRNAs are relocalized to P-bodies ina Bfr1p-dependent manner.

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mRNA is also a component of these granules and there is no reasonto suspect that the mRNA would rapidly exit, or become degraded

in a stress granule, as the mRNA decay machinery is absent.Therefore, we favor an explanation where only specific mRNAsare localized, although an example of such an mRNA has yet to beidentified.

The formation of P-bodies provides an opportunity for cells totarget their mRNA content for mRNA decay and/or storage. Thefact that individual mRNAs are recruited to these granules in

distinct phases, which have distinct cofactor requirements,suggests that this process is more complicated than previouslyanticipated. Ultimately, this high degree of fine-tuning, in terms of

the regulation of mRNA movement to granules, suggests that cellsfollow a precisely determined program of reorganization afterstress. It is possible that each facet of this reorganization process

would hold an evolutionary advantage in terms of cell survival.

MATERIALS AND METHODSStrains and plasmidsS. cerevisiae strains used in this study are listed in supplementary

material Table S2. Proteins were tagged at the C-terminus and verified by

PCR analysis (Campbell and Ashe, 2007). MS2-binding sites were

inserted into 39UTRs and verified using PCR and RT-PCR (Fig. 1) (Haim

et al., 2007). Knockout strains were generated using a KanMX2 insertion

cassette (Wach et al., 1994) and verified using PCR on genomic DNA

samples. The edc3D lsm4DC mutant (kindly provided by J. Hasek)

(Grousl et al., 2009) was backcrossed four times to W303-1A, then

backcrossed to the MS2L-tagged strains to generate edc3D lsm4DC

DCP2-CFP MS2L-mRNA strains.

Growth conditionsCells were grown at 30 C to OD600 0.4 in synthetic complete medium

with 2% glucose (SCD) (Guthrie and Fink, 1991). Cells were incubated

for 1 hour in SCD medium lacking methionine to induce expression of

pCP-GFP3. For stress conditions, cells were incubated in medium lacking

glucose (SC) for 10 minutes or 50 minutes.

Microscopy and quantificationEpifluorescent images used for quantification were acquired on an

Eclipse E600 microscope using a 1006/1.40 numerical aperture oil plan

Apo objective. Images were collected using Axiovision 4.5 software

(Carl Zeiss MicroImaging, Inc.) and camera. Representative cells are

shown from experiments repeated at least three times. Granules were

counted using 100 cells for each mRNA in triplicate. All other images

were taken using the delta vision RT (Applied Precision) with a 1006/

1.40 numerical aperture differential inference contrast oil plan Apo

objective (Olympus) and camera (CoolSNAP HQ; Roper Scientific)

using Softworx 1.1 software (Applied Precision) and 161 binning.

Quantitative real-time reverse transcriptase PCRRNA analysis by quantitative reverse transcription PCR (qRT-PCR) was

carried out using the iScriptTM one-step RT-PCR kit with SYBRH green

(Bio-Rad) on a CFX connectTM real-time PCR detection system (Bio-

Rad). The primers used are listed in supplementary material Table S3 and

signals were quantified relative to actin mRNA using the 22DDCt method

(Livak and Schmittgen, 2001).

Protein analysisTAP tag purification experiments were completed using metal tosyl-

activated dynabeads (Invitrogen) bound to 10 mg/ml IgG. 10 mg of total

protein prepared from exponentially growing yeast cells was incubated

with the beads for 20 minutes. Where samples were RNase treated, 100 ml

RNase I (Ambion) was added during the incubation with beads. Beads

were washed five times in buffer (20 mM Tris-HCl pH 8, 140 mM NaCl,

1 mM MgCl2, 0.5% NP-40, 0.5 mM DTT, 1 mM phenylmethylsulfonyl

fluoride). Bound protein was analyzed by western blotting. TAP-tagged

proteins were detected using horseradish peroxidase (HRP)-conjugated

protein A (PAP) (Abcam), or 96Myc proteins were detected using a Myc

antibody (Millipore) and Tef1p was detected using an endogenous

antibody (a gift from Chris Grant).

Proteomic analysesCells were grown at 30 C to an OD600 of 0.8 and depleted of glucose for

50 minutes, as for the microscopy and western blotting analysis. Bound

proteins were eluted from the IgG DynabeadsH (Invitrogen) using

sequential solutions of 0.5 M acetic acid then 500 mM hydrogen

peroxide. Whole eluates were dried down and resuspended in a solution

of 80% acetonitrile, 20% 50 mM Tris-HCl pH 7.6, 10 mM CaCl2 and

250 ng trypsin. Samples were incubated for 1 hour at 37 C, then dried

down and resuspended in 10% acetonitrile and 0.1% formic acid for mass

spectrometry analysis. Mass spectrometry analysis was performed using

the SYNAPT HDMSTM (Waters) mass spectrometer followed by analysis

using PGLS analysis software.

AcknowledgementsWe thank J. Gerst, Chris Grant and J. Hasek for kindly providing reagents and H.Ashe for critique of the manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsC.E.S. performed most of the experiments in the paper and contributed to thewriting process. J.L. performed some of the qRT-PCR analysis, the detailed timecourse and some of the bfr1 mutant analysis. C.J.K. performed theimmunopreciptations. M.P.A. led and coordinated the study and wrote the paper.All authors contributed intellectually to the experimental design, interpretation ofthe data and approved the manuscript.

FundingThis work was largely supported by the Wellcome Trust [grant number 088141/Z/09/Z]. J.L. was supported by a Biotechnology and Biological Sciences ResearchCouncil (BBSRC) studentship, and project grant [number BB/K005979/1] andC.J.K. was supported by a LoLa BBSRC grant [grant number BB/G012571/1].Deposited in PMC for immediate release.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139055/-/DC1

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