Decapping of stabilized, polyadenylated mRNA in yeastpab1 mutants

Yeast 15, 687–702 (1999)

Decapping of Stabilized, Polyadenylated mRNA inYeast pab1 Mutants

JOHN P. MORRISSEY2, JULIE A. DEARDORFF1, CLARISSA HEBRON1 AND ALAN B. SACHS1*1Department of Molecular and Cell Biology, 401 Barker Hall, University of California at Berkeley, Berkeley,CA 94720, U.S.A.2Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH, U.K.

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. Thiswork describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to besensitive to elevated concentrations of copper (Cu) and 3-aminotriazole (3-AT) in the growth medium. Thisphenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, theproducts of which are required for Cu and 3-AT resistance, respectively. To determine the cause of the mRNAunderaccumulation, mRNA turnover and production were examined in the pab1-53 mutant. It was found thatalthough the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could bedetected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in theproduction of mRNA. These data show that the decreased level of mRNA in the pab1-53 mutant arises from poorproduction, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They alsoindicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA’s stability,and that this uncoupling can be tolerated by yeast. Copyright ? 1999 John Wiley & Sons, Ltd.

— mRNA degradation; mRNA processing; PAB1; poly(A); yeast

1990). Experiments using a yeast-derived in vitro

*Correspondence to: Dr Alan Sachs, Department of Molecularand Cell Biology, 401 Barker Hall, UC Berkeley, CA 94720,U.S.A. e-mail: [email protected]/grant sponsor: American Cancer Society; Contract/grant number: 82666.Contract/grant sponsor: National Institute of Health; Contract/grant number: R01-GM50308.Contract/grant sponsor: Hellman Family Fund.

INTRODUCTION

Eukaryotic mRNA undergoes a number ofprocessing and modification reactions beforeexport from the nucleus. In a process that is closelycoupled to transcription, mRNA acquires a modi-fied nucleotide at the 5* end, the m7G cap. Proteinswhich interact with this cap are important forsplicing, transport and translation of the mRNA.Many mRNAs are spliced to remove introns, andall mRNAs, with the exception of some histonemRNAs in higher eukaryotes, receive a 3* poly-adenylate tail. Many of the factors involved inpolyadenylation have been identified, initially bio-chemically in higher eukaryotes, and more recentlyby a combination of biochemistry and genetics in

CCC 0749–503X/99/080687–16 $17.50Copyright ? 1999 John Wiley & Sons, Ltd.

Saccharomyces cerevisiae (reviewed in Keller andMinvielle-Sebastia, 1997). The cleavage and poly-adenylation complex assembles on the 3* untrans-lated region of the mRNA, cleaves the RNA andadds a poly(A) tail, the length of which varies from70 residues in yeast to approximately 200 residuesin higher eukaryotes.

It is not yet clear whether the poly(A) tail has anuclear function, e.g. in facilitating export ofmRNA, but a cytoplasmic role for the poly(A) tailhas been established. The highly conservedpoly(A) binding protein, Pab1p, is believed to bethe primary mediator of poly(A) tail function inthe cytoplasm. Data from a number of systemssuggest that the poly(A) tail is involved in thetranslation of mRNA. Expression of some mater-nal mRNAs during embryonic development istranslationally regulated by cytoplasmic adenyla-tion and deadenylation of the mRNA (reviewed inCurtis et al., 1995). In vitro, the poly(A) tail isrequired for efficient translation (Gallie, 1991;Iizuka et al., 1994; Munroe and Jacobson,

Received 21 September 1998Accepted 2 January 1999

688 J. P. MORRISSEY ET AL.

translation system have confirmed the importanceof the poly(A) tail for translation and have deter-mined that, through its interaction with Pab1p, thepoly(A) tail stimulates joining of the 40S ribo-somal subunit at the 5* end of the mRNA (Tarunand Sachs, 1995). This step of translation initiationis also stimulated by the 5* cap through an associ-ation of the cap-binding protein eIF4E with thetranslation initiation factor eIF4G (Hershey et al.,1996). Poly(A)-bound Pab1p also interacts witheIF4G, leading to the model that the 5* cap and the3* poly(A) tail act in concert to bind eIF4G,ultimately recruiting the 40S ribosomal subunit tothe 5* end of the mRNA (Tarun and Sachs, 1996;Tarun et al., 1997; Hentze, 1997; Sachs et al., 1997;Kessler and Sachs, 1998).

This complex between the 5* and 3* ends of themRNA has been proposed to play a second role inthe cell, namely, protection of the 5* cap fromnucleolytic attack (reviewed in Caponigro andParker, 1996). It has been demonstrated that deg-radation of many yeast mRNAs follows a sequen-tial pattern, starting with removal of the poly(A)tail, proceeding with removal of the m7G cap atthe 5* end of the mRNA and culminating withdegradation of the body of the mRNA by a 5*–3*exonuclease. Some of the factors involved in theseprocesses have been identified: removal of the capis carried out by the Dcp1p protein, the activity ofwhich may be regulated by the products of theMRT1, MRT3 and SPB8 genes (Beelman et al.,1996; Hatfield et al., 1996; Lagrandeur and Parker,1998; Boeck et al., 1998). The major 5*–3* exonu-clease responsible for degradation of the messageis Xrn1p (Muhlrad et al., 1994).

The precise rates of mRNA deadenylation anddecapping appear to be intrinsic properties of anyparticular mRNA and, as such, both impact onmRNA half-lives. As outlined above, deadenyla-tion ordinarily precedes decapping, but it wasfound that a yeast strain carrying a null allele ofPAB1, viable because of a bypass suppressor,decapped mRNA which was still polyadenylated(Caponigro and Parker, 1995). This finding led tothe model that a primary function of the poly(A)tail is to protect the 5* end of the mRNA. It wasproposed that, in the absence of Pab1p, the 5* capwas prematurely removed, thereby causing mRNAto be prematurely degraded. A tenet of this modelis that premature decapping is a lethal phenotypedue to premature degradation of the mRNA. Thatdeletion of XRN1 allowed yeast cells to grow with-out Pab1p provided strong support for this model.

Copyright ? 1999 John Wiley & Sons, Ltd.

The work described in this paper deals withfurther characterization of the role of Pab1p in theyeast cell. Strains carrying mutant alleles of PAB1were found to underaccumulate mRNA. Althoughpolyadenylated mRNA was decapped in thesestrains, mRNA was not less stable. Instead, thelower levels of mRNA could be accounted for by areduced rate of production. The implications ofthis finding for a nuclear function for Pab1p arediscussed.

MATERIALS AND METHODS

Strains and plasmidsYeast strains constructed for this study are listed

in Table 1 and plasmids are listed in Table 2. Adiagram of the various pab1 alleles is shown inFigure 1. The parent strain for all the strainscarrying the pab1 disruption is a W303 derivative,YAS307 (MATá ade2-1 his3-11 leu2-3,112 trp1-1ura3-1 can1-100). All the strains listed in Table 2are MATá ade2 his3 leu2 trp1 ura3. Thexrn1::LEU2 allele (source: S. Peltz) and the rpb1-1allele (source: R. Young) were backcrossed intothe YAS307 genetic background, and the progenyof those crosses used to derive YAS2199, YAS2205and YAS2206. Standard media, growth conditionsand techniques for handling yeast were used(Guthrie and Fink, 1991). Where indicated, Cu, inthe form of CuSO4, or 3-amino-1,2,4-triazole (3-AT) (Sigma) was added to the growth media at thestated concentrations.

Analysis of GCN4-lacZ reportersA construct, p180, consisting of the GCN4 5*

UTR fused to the lacZ ORF (Hinnebusch, 1985)was introduced into yeast strains. To assess theability of various mutants to induce its expression,yeast were grown in minimal media to mid-log andthen split into two cultures. One culture was made20 m 3-aminotriazole (3-AT), and then both cul-tures were grown for another 6 h. The level of lacZproduction was quantified by B-galactosidase as-says (Miller, 1972). Briefly, 1 OD600 of cells werepelleted and resuspended in 1 ml Z-buffer (60 mNa2HPO4, 40 m NaH2PO4, 10 m KCl, 1 mMgSO4). Cells were lysed by vortexing for 10 safter adding 3 drops of CHCl3 and 2 drops of 0·1%SDS. 100 ìl of lysate were incubated with 134 ìlof Z buffer and 66 ìl of ONPG (4 mg/ml stock).The incubation was continued at 37)C until ayellow colour was apparent, or for 30 min, and the

Yeast 15, 687–702 (1999)

689UNDERACCUMULATION OF mRNA IN PAB1 MUTANTS

reaction was quenched by the addition of 500 ìlNaHCO2. The OD420 was recorded and the unitsof enzyme activity calculated as described (Miller,1972).

Table 1. Yeast strains constructed in this work.

Strain Relevant genotype

YAS2170 pab1::HIS3 [pab1-52 TRP1 CEN]YAS2171 pab1::HIS3 [pab1-53 TRP1 CEN]YAS2172 pab1::HIS3 [PAB1 TRP1 CEN]YAS2173 pab1::HIS3 [pab1-55 TRP1 CEN]YAS2178 pab1::HIS3 [pab1-53 TRP1 CEN] [URA3-2m]YAS2179 pab1::HIS3 [pab1-53 TRP1 CEN] [CUP1 URA3 2m]YAS2180 pab1::HIS3 [pab1-53 TRP1 CEN] [HIS3 URA3 2m]YAS2181 pab1::HIS3 [PAB1 TRP1 CEN] [CGN4::lacZ URA3 CEN]YAS2182 pab1::HIS3 [pab1-53 TRP1 CEN] [CGN4::lacZ URA3 CEN]YAS2183 pab1::HIS3 [pab1-52 TRP1 CEN] [CGN4::lacZ URA3 CEN]YAS2184 pab1::HIS3 [pab1-55 TRP1 CEN] [CGN4::lacZ URA3 CEN]YAS2187 pab1::HIS3 [PAB1 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2188 pab1::HIS3 [pab1-38 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2189 pab1::HIS3 [pab1-52 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2190 pab1::HIS3 [pab1-53 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2191 pab1::HIS3 [pab1-66 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2192 pab1::HIS3 [pab1-55 TRP1 CEN] [GAL::MFA2pG URA3 CEN]YAS2193 pab1::HIS3 [PAB1 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2194 pab1::HIS3 [pab1-38 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2195 pab1::HIS3 [pab1-52 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2196 pab1::HIS3 [pab1-53 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2197 pab1::HIS3 [pab1-66 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2198 pab1::HIS3 [pab1-55 TRP1 CEN] [GAL::PGK1pG URA3 CEN]YAS2199 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN]YAS2200 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN] [PAB1-38 TRP1 CEN]YAS2201 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN] [PAB1-52 TRP1 CEN]YAS2202 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN] [PAB1-53 TRP1 CEN]YAS2203 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN] [PAB1-66 TRP1 CEN]YAS2204 pab1::HIS3 xrn1::LEU2 [PAB1 URA3 CEN] [PAB1-55 TRP1 CEN]YAS2205 pab1::HIS3 rpb1-1 [PAB1 TRP1 CEN]YAS2206 pab1::HIS3 rpb1-1 [pab1-53 TRP1 CEN]

Transcriptional shut-off methodsFor experiments involving glucose repression of

transcription, yeast strains containing either thepRP485 (Decker and Parker, 1993) or pRP602(Muhlrad et al., 1995) reporters were grown at25)C to mid-log phase in 400 ml of YMGALsupplemented with the appropriate nutrients. Cellswere pelleted, washed, and resuspended in 20 ml ofYM media containing 4% glucose. Aliquots(1·5 ml) were withdrawn at fixed time intervals, thecells were pelleted by microcentrifugation, and thepellets were quick-frozen in liquid nitrogen. RNAwas extracted from the pellets as described below.


For the transcriptional pulse-chase experiments,yeast were grown in 500 ml cultures of YMDmedium at 25)C to an OD600 of approximately 0·4.Cells were concentrated by centrifugation andresuspended in 20 ml YMD. CuSO4 was added toa final concentration of 0·5 m. The cells wereincubated at 25)C for 8 min, centrifuged, washedwith YMD to remove CuSO4, and resuspended in10 ml YMD at 25)C. 10 ml of preheated YMD(58)C) were added so that the temperature of thecells was immediately brought to 37)C. The totaltime between addition of CuSO4 and the cellsbeing brought to 37)C was 12 min. The cells wereincubated at 37)C and 1·5 ml aliquots of cells werecollected at defined times, pelleted and quick-frozen in liquid nitrogen. Aliquots were alsocollected before the addition of Cu (uninducedtime-point) and before the temperature shift (0 min

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time-point). RNA was extracted from the pellets asdescribed below.

RNA methodsRNA was recovered from frozen cell pellets by

resuspending them in 800 ìl extraction buffer(300 m NaCl, 20 m Tris–HCl pH 7·4, 10 mEDTA, 1% SDS). 500 ìl phenol, preheated to65)C, were added and the suspension was incu-bated at 65)C for 4 min with occasional vortexingon a multi-tube vortexer. The suspension waschilled on ice and the phases separated by centrifu-gation at 4)C. The aqueous phase was re-extractedwith phenol, with phenol–CHCl3, and with CHCl3.The RNA was precipitated with 2 volumes ofethanol.

RNase H was used to remove poly(A) tails frommRNA. 10 ìg total yeast RNA were incubatedwith 300 ng oligo(dT) (Pharmacia) and 0·2 unitsRNase H (GibcoBRL) in reaction buffer provided


by the supplier for 30 min at 30)C. The reactionwas stopped by the addition of an equal volume of2# PAGE gel loading buffer (95% formamide,20 m EDTA).

Table 2. Plasmids used in this study.

Plasmid Genotype Source

pAS77 [PAB1 URA3 CEN] Sachs et al. (1987)pAS80 [PAB1 TRP1 CEN] Sachs et al. (1987)pBAS78 [pab1-38 TRP1 CEN] Sachs et al. (1987)pBAS83 [pab1-52 TRP1 CEN] Sachs et al. (1987)pBAS89 [pab1-53 TRP1 CEN] Sachs et al. (1987)pBAS75 [pab1-66 TRP1 CEN] Sachs et al. (1987)pAS401 [pab1-55 TRP1 CEN] Sachs et al. (1987)yEPlac195 [URA3 2ì] Gietz & Sugino (1988)pAS563 [CUP1 URA3 2ì] This studypAS564 [HIS3 URA3 2ì] This studyp180 [GCN4::lacZ URA3 CEN] Hinnebusch et al. (1985)pRP485 [GAL::MFA2pG URA3 CEN] Decker et al. (1993)pRP602 [GAL::PGK1pG URA3 CEN] Muhlrad et al. (1993)

Figure 1. PAB1 alleles used in this study. The structure ofPAB1 is shown schematically with the location of the fourRNA recognition motifs (RRMs) indicated. The mutant pab1alleles are also shown, with the hatching denoting deletedregions.

Northern blotting and hybridization methodsRNA was separated either by polyacrylamide

gel electrophoresis (PAGE) or by agarose gel elec-trophoresis. For PAGE, approximately 10 ìg ofRNA in PAGE loading buffer were loaded on to0·75 mm thick 6% polyacrylamide, 8·3 Urea,0·5# TBE gels, which were then run for 2000–4000 volt hours, depending on the RNA to bevisualized. RNA was transferred to Zetaprobemembranes (Bio-Rad) by electroblotting.

For agarose gel electrophoresis, approximately10 ìg of RNA in agarose gel loading buffer (50%formamide, 6% formaldehyde, 1# running buffer)were loaded on to 1·2% agarose, 6% formaldehyde,1# running buffer gels and electrophoresed inrunning buffer (20 m MOPS, 8 m NaAc,1 m EDTA). RNA was transferred to Zetaprobemembranes by capillary transfer.

Zetaprobe membranes were hybridized in 7%SDS, 250 m NaPO4, pH 7·2, 2 m EDTA, asdescribed by the manufacturer. 20 ng of oligo-nucleotide were 32P-labelled at the 3* end withterminal deoxytransferase, and hybridizationcarried out at 50)C. 100 ng of DNA template,derived either from a PCR reactoin or from aplasmid, were 32P-labelled using random primersand the Klenow fragment of DNA polymerase,and hybridization carried out at 65)C. Washingwas as recommended by the manufacturer, at 50)C

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for oligonucleotide probes, and at 65)C for ran-domly primed probes.

RESULTS

pab1 mutants are sensitive to high concentrationsof copper and 3-aminotriazole

The poly(A) binding protein, Pab1p, is highlyconserved across eukaryotes, with the greatestconservation being in the four N-terminal RNArecognition motifs (RRMs). These RRMs arerequired for interaction between Pab1p and RNA(Sachs et al., 1987; Nietfeld et al., 1990; Burd et al.,1991; Kuhn and Pieler, 1996; Deardorff and Sachs,1997) as well as for the interaction between Pab1pand eIF4G (Kessler and Sachs, 1998). Previousstudies in our laboratory generated deletion muta-tions in PAB1, some of which are shown schemati-cally in Figure 1 (Sachs et al., 1987). Although allthe pab1 alleles depicted here supported growth,it has now been observed that strains carryingcertain of these alleles are sensitive to elevatedconcentrations of copper (Cu) or 3-aminotriazole

Figure 2. Mutations in PAB1 cause sensitivity to copper and 3-aminotriazole. (A) Yeast strains YAS2172(PAB1), YAS2170 (pab1-52), YAS2171 (pab1-53) and YAS2173 (pab1-55) were streaked on minimal plates(YMD) or minimal plates supplemented with 75 m 3-aminotriazole (3-AT) or 0·5 m CuSO4 as indicated,and incubated at 30)C for 3 days. (B) Yeast strains YAS2171 (pab1-53) was transformed with an empty highcopy 2 ì vector, or with that vector carrying the CUP1 or HIS3 gene, to create strains YAS2178, YAS2179and YAS2180, respectively. These strains were plated on minimal media, or on minimal media supple-mented with 75 m 3-AT or 0·75 m CuSO4, and incubated at 30)C for 3 days (YMD and YMD+3-AT)or 5 days (YMD+CuSO4).


(3-AT) in the growth medium (Figure 2A). Onlydeletions within the RRM domains of the proteingive rise to Cu/3-AT sensitivity, suggesting thatimpaired interaction with the poly(A) tail may beresponsible for the phenotype.

The Cu sensitivity of the pab1 mutants could bedue to a reduced amount of the Cup1 protein. Inyeast, excess Cu is chelated by a metallothionein,Cup1p, preventing its accumulation to toxic levels.When extracellular Cu concentrations are low, theCUP1 gene is transcribed at a basal level. Yeastrespond to high concentrations of Cu by inducingtranscription of CUP1 via the transcriptional acti-vator Ace1p (reviewed in Tohoyama et al., 1995).It has previously been demonstrated that thedegree of sensitivity/resistance to Cu correlateswith the level of Cup1 protein in the cell (Lesserand Guthrie, 1993).

In a similar manner, the 3-AT sensitivity of thepab1 mutants could result from lower levels of theHis3 protein. 3-AT is a competitive inhibitor ofHis3p, a histidine biosynthetic enzyme. Treatingyeast with this drug inhibits the synthesis of histi-dine and activates the amino acid starvation

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Table 3. Induction of GCN4-lacZ in pab1 mutants.

AlleleRelative level

(%)Induction

(fold)

PAB1 100 1·9&0·4pab1-52 90&6 2·6&0·5pab1-53 71&11 3·0&1·1pab1-55 104&14 1·9&0·3

Strains YAS2181 (PAB1), YAS2183 (pab1-52), YAS2182(pab1-53) and YAS2184 (pab1-55), carrying the GCN4–lacZreporter construct p180, were grown in minimal media tomid-log phase. They were then incubated at 30)C for 6 h in thepresence or absence of 20 m 3-aminotriazole (3-AT). Cellswere harvested and B-galactosidase assays carried out to deter-mine the level of the lacZ protein. Relative level refers to thelevel of lacZ protein before treatment with 3-AT with the levelin YAS2181 taken as 100%. The fold induction was calculatedby dividing the lacZp level after induction by the level beforeinduction for each strain. The values shown are average valuesderived from two (pab1-52, pab1-55) or three (PAB1, pab1-53)independent experiments.

response pathway (reviewed in Hinnebusch, 1992).An early step in this pathway is the synthesis of theGcn4p transcription factor, which then activatestranscription of amino acid biosynthetic genes,including HIS3, and ultimately leads to theupregulation of amino acid biosynthesis.

To test whether Cup1p or His3p were limiting inthe pab1-53 mutant, the CUP1 and HIS3 geneswere cloned into high copy number plasmids andtransformed into the mutant. As shown in Figure2B, the overexpression of these genes led to theloss of both the Cu and 3-AT sensitivities. Theseresults are consistent with the hypothesis that thesesensitivities arose from underaccumulation of theCUP1 and HIS3 gene products in this strain.

Regulated Gcn4p translation, and acidicactivator-dependent transcription, are intact inpab1 mutant yeast strains

Since Pab1p has been implicated in translationalcontrol, it was investigated whether the 3-ATsensitivity of the pab1 mutants arose from a trans-lational defect. This possibility was attractive sinceexpression of GCN4 is controlled at the level oftranslation, and mutations in some translationfactors have been shown to alter 3-AT sensitivity/resistance. Translation of GCN4 mRNA is regu-lated by short open reading frames (uORFs) in the5-UTR of the mRNA. Under non-starvation con-ditions, these uORFs act to repress translation ofthe GCN4 ORF, whereas under amino acid-limiting conditions translation of GCN4 is dere-pressed and protein synthesis ensues (Hinnebusch,1992).

A reporter construct comprising the 5* UTR ofGCN4 fused to the lacZ coding sequence was usedto monitor GCN4 expression in yeast strains(Hinnebusch, 1985). When cells harbouring thisreporter are grown on complete medium, transla-tion of lacZ mRNA is repressed by the GCN4 5*UTR. Treating cells with 3-AT activates the aminoacid starvation response and induces translation ofthe lacZ reporter. Yeast strains carrying themutated pab1 alleles and the reporter constructwere grown for 6 h in minimal medium in thepresence or absence of 20 m 3-AT. The cells werethen harvested and the lacZ protein levels quanti-fied by B-galactosidase assays. The results aresummarized in Table 3. Even though the initiallevel of lacZ was lower in some of the pab1 mutantstrains, all the strains induced translation of thereporter construct in response to 3-AT. The strong

induction in the mutants indicate that the 3-ATsensitivity is not a consequence of an inability toupregulate translation of the GCN4 mRNA.

Resistance to Cu and 3-AT requires the activityof the acidic transcriptional activators Ace1p andGcn4p, respectively. Acidic activators functionthrough an interaction with the basal transcriptionfactors, TBP and TFIIA. It has been shown thatmutants defective in the TBP–TFIIA interactioncannot induce transcription with acidic activatorsand, as a result, are sensitive to Cu and 3-AT (Leeand Struhl, 1995; Stargell and Struhl, 1995). Thesemutants are also unable to utilize galactose as acarbon source, since expression of the galactose-metabolizing genes requires the function of theacidic activator Gal4p. These observations led usto investigate whether sensitive pab1 mutants wereable to induce transcription of the CUP1 and HIS3genes on treatment with Cu and 3-AT, respect-ively. Cu-sensitive strains carrying the pab1-52 orpab1-53 allele, and Cu-resistant strains carryingthe PAB1 or pab1-55 allele, were treated withincreasing concentrations of Cu for 1 h (Figure3A). All strains showed a strong induction ofCUP1 mRNA over the level present in the unin-duced cells. Similarly, the same strains treated with3-AT for 6 h induced transcription of the HIS3gene (Figure 3B). In addition, all of these strainswere able to utilize galactose as a carbon source,indicating that the acidic activator Gal4p is func-tional (data not shown). These data show that

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Figure 3. pab1 mutants can induce expression of CUP1 andHIS3. Strains YAS2172 (PAB1), YAS2170 (pab1-52), YAS2171(pab1-53) and YAS2173 (pab1-55) were grown on minimalmedia to mid-log phase and then treated with CuSO4 or3-aminotriazole (3-AT). (A) Cells were incubated with thefollowing CuSO4 concentrations for 1 h: lanes 1, 4, 7 and 10, noCuSO4; lanes 2, 5, 8 and 11, 0·25 m CuSO4; lanes 3, 6, 9 and12, 1 m CuSO4. RNA recovered from the cells was separatedon a formaldehyde–agarose gel and detected by hybridizationwith the indicated probes (CUP1 or SCR1). (B) Cells wereincubated for 6 h without (lanes 1, 2, 3 and 4) or with (lanes 5,6, 7 and 8) 10 m 3-aminotriazole. RNA was recovered fromthe cells, separated on a formaldehyde–agarose gel, anddetected by hybridization with the indicated probes (HIS3 orSCR1).

is consistent with this interpretation.


Figure 4. mRNA underaccumulates in pab1 mutants. RNAwas recovered from strains YAS2172 (PAB1), YAS2170 (pab1-52), YAS2171 (pab1-53) and YAS2173 (pab1-55) grown in YMmedium, separated on a 6% polyacrylamide gel and detected byhybridization with the following probes: HIS3, CUP1, HTB1(encodes the histone H2B protein), CYH2 and RPL46 (encoderibosomal proteins), and SCR1.

the observed Cu and 3-AT sensitivities in pab1mutants do not result from loss of acidic activatorfunction.

pab1 mutants have reduced levels of mRNAAlthough pab1 mutants induced transcription of

the CUP1 and HIS3 genes, it was observed that,compared to the wild-type (wt) strain, the absolutelevels of mRNA were lower in the mutants. Boththe basal mRNA levels and the mRNA levelsfollowing induction were reduced (Figure 3). Thus,it seems likely that the Cu and 3-AT sensitivities ofthe pab1 mutants are due to lower levels of theCUP1 and HIS3 mRNAs. The suppression of thesensitivity phenotypes by overexpression of CUP1and HIS3 from their native promoters (Figure 2B)

In these experiments, approximately equalquantities of total RNA, the bulk of which com-prises rRNA, were loaded on the gels. The stableRNA Polymerase III (Pol III) transcript SCR1,which encodes the RNA component of the signalrecognition particle, was used as a loading controlto allow direct comparisons between samples.Although strains carrying the pab1-52 and pab1-53alleles grow more slowly than wt strains, therehave not been any reports that slow growth ratescause a reduction in the level of RNA polymeraseII (Pol II) transcripts relative to those producedby RNA polymerases I and III. The questiontherefore arose as to whether this effect of reducedmRNA levels in the pab1 mutants was peculiar toCUP1 and HIS3, or whether it was a more generalphenomenon. To address this, the levels of anumber of mRNAs in the pab1 mutant strainswere assessed by Northern analysis (Figure 4).mRNA is visualized as a broad band by thistechnique, since the high resolution allows thedetection of mRNA with the gamut of poly(A) taillengths. In this experiment, SCR1 was again usedas a loading control. Notwithstanding slight
underloading of the pab1-52 and pab1-53 lanes,
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indicated by the levels of the SCR1, it was foundthat the levels of all mRNAs examined werereduced in the mutant strains. The extent of thereduction was message-specific but typicallymRNAs were reduced to between 30% and 50% ofthe wt level. These data are indicative of a generaldefect in the accumulation of mRNA in pab1mutants rather than a specific defect for CUP1 orHIS3 mRNA.

A deletion of XRN1 exhibits synthetic lethalinteractions with pab1 mutations

In addition to translation, Pab1p has beenimplicated in the process of mRNA turnover.Pab1p binds to the poly(A) tail of mRNA in thecytoplasm and it has been proposed that an inter-action between Pab1p bound to the poly(A) tailand a complex at the 5* end of the mRNA protectsthe 5* cap from degradation. In this model,removal of the poly(A) tail by a deadenylase woulddisrupt the complex at the 5* end, thereby exposingthe cap to the Dcp1p decapping enzyme. Afterdecapping, the Xrn1p exonuclease destroys thebody of the mRNA.

Deletion of the XRN1 gene, encoding the majorcytoplasmic 5*–3* exonuclease, stabilizes mRNAwhich has been decapped (Hsu and Stevens, 1993).It was previously reported that a strain lackingXrn1p was viable in the complete absence ofPab1p (Caponigro and Parker, 1995). Becauseunderaccumulation of mRNA was the probablecause of the observed sensitivities of the pab1mutants, whether deletion of the XRN1 gene inthese mutant strains would lead to suppression oftheir sensitivities was investigated. Accordingly, astrain was constructed which carried deletions inboth the XRN1 and PAB1 genes, and which reliedon a plasmid-borne wt allele of PAB1 for survival.Mutant alleles of pab1 were introduced into thisstrain, and a plasmid shuffle technique was used toeliminate the wt PAB1 allele from the cell. Surpris-ingly, it was found that a deletion of XRN1 did notsuppress a PAB1 deletion (Figure 5). This is incontradiction to the previous report (Caponigraand Parker, 1995) and cannot readily be explainedby strain differences since our crosses using strainsprovided by those researchers, yRP919 andyRP920, also failed to show xrn1 suppression ofa pab1 deletion. At this time, the discrepancyremains unresolved. Furthermore, it was observedthat the pab1-52, pab1-53 and pab1-66 alleles couldno longer support growth when the XRN1 gene


was deleted. In contrast, the pab1-38 and pab1-55alleles still supported growth in the absence ofXrn1p (Figure 5). The inability of the pab1-53allele to support growth in this strain could berescued by the introduction of a wt allele of XRN1on a plasmid, confirming that the syntheticlethality was due to deletion of the XRN1 gene(Figure 5B).

The pab1-53 mutation leads to mRNAstabilization

The observation that mRNA levels were lowerin pab1 mutant strains, in conjunction with theprevious report that a PAB1 deletion promotesmRNA instability (Caponigro and Parker, 1995),led us to examine mRNA turnover in pab1 mutantstrains. A strain carrying the pab1-53 allele waschosen for these experiments, since strains carryingthis pab1 allele displayed the most severe Cu/3-ATsensitivities and the lowest mRNA levels. The ratesof mRNA decay were compared in strains carryingthe pab1-53 allele and in otherwise isogenic strainswhich carried a wild-type PAB1 allele. The resultsare summarized in Table 4.

Initially, degradation of the well-characterizedMFA2 mRNA was examined. A reporter con-struct, comprising the MFA2 coding sequenceunder the control of a regulatable GAL promoter(Decker and Parker, 1993), was introduced intostrains carrying the wt and pab1-53 alleles. Cellswere grown in medium with galactose as thecarbon source, allowing expression of the MFA2mRNA. Glucose was then added to repress tran-scription of the MFA2 gene. The levels of MFA2mRNA were assessed by Northern blotting beforethe addition of glucose (Figure 6A, lanes 1 and 7)and at various times afterwards (Figure 6A, lanes2–6 and 8–14) and were plotted as a function oftime (Figure 6B). The addition of glucose causedrapid transcriptional shut-off, allowing the half-lifeof MFA2 mRNA to be calculated from the slopeof the decay curve. In the strain carrying the wtPAB1 allele, the MFA2 mRNA had a half-life of3·0 min, which is in good agreement with thepreviously determined half-life of MFA2 mRNA(Herrick et al., 1990; Decker and Parker, 1993).In the pab1-53 mutant strain, however, MFA2mRNA decayed with a half-life of 7·3 min. MFA2mRNA is therefore more rather than less stable inthe pab1-53 mutant.

To investigate whether the unexpected stabiliz-ation of mRNA was a general phenomenon, a

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Figure 5. A deletion of XRN1 is synthetically lethal with mutations in PAB1. (A) Yeaststrain YAS2199, which is deleted in the genome for XRN1 and PAB1 and carries a wtallele of PAB1 on a URA3 CEN plasmid (pAS77), was transformed with the indicatedTRP1 CEN plasmids carrying mutant pab1 alleles. These strains were plated on minimalmedia (YMD) or on minimal media supplemented with 5-FOA, which only allows for thegrowth of cells which have lost the PAB1 URA3 CEN plasmid. The plates were incubatedat 30)C for 3 days (YMD) or 5 days (YMD+5-FOA). Even after long incubation of theYMD+5-FOA plate (10 days), no growth of strains carrying the pab1-66, pab1-53 orpab1-52 alleles was observed. (B) Strains YAS2199 was transformed with plasmidscarrying the indicated alleles and transformants were plated on YMD or on YMD+5-FOA to force the loss of the PAB1 URA3 CEN plasmid. pab1-53 could only supportgrowth in the presence of XRN1.

Table 4. mRNA half-lives (minutes) in PAB1 andpab1-53 strains.

mRNA PAB1 pab1-53

MFA2 3·0&0·1 7·3&1·2CUP1 13&1·8 21&6·9PGK1 22&1·0 47&5·0

mRNA half-lives were calculated as described in Figures 6, 7and 8. The values shown are an average of two (MFA2, PGK1)or three (CUP1) independent experiments.

similar transcription shut-off experiment was per-formed with the yeast PGK1 gene fused to theGAL promoter (Figure 7). As with MFA2, PGK1was stabilized approximately two-fold in thepab1-53 mutant (Table 4) demonstrating thatstabilization of mRNA in the pab1-53 mutant isnot limited to unstable mRNAs such as MFA2 butalso extends to stable mRNA.


It was previously reported that, in a pab1 nullstrain, newly synthesized mRNA underwent a lagof 15–20 min before decaying (Caponigro andParker, 1995). This lag represents a phase wheremRNA has not yet entered the decay pathway. Wehave also observed this lag in pab1 null strains.The experiments described above measured steady-state mRNA decay rates which would be distortedby a lag and it was therefore necessary to examinethe decay of newly transcribed mRNA. This hasbeen previously accomplished by manipulation ofexpression from the GAL promoter: transfer ofcells grown on raffinose to galactose led to a rapidpulse of transcription from the GAL promoter(Decker and Parker, 1993).

Poor growth on raffinose and slow induction ontransfer to galactose meant that this approach wasnot possible in our strains, so an alternativemethod was devised. The rapid induction of theCUP1 gene on treatment with Cu allowed theCUP1 mRNA to be developed as a reporter for

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Figure 6. Decay of MFA2 mRNA in PAB1 and pab1-53strains. (A) Yeast strains YAS2187 (PAB1) and YAS2190(pab1-53), carrying the GAL::MFA2 reporter (pRP485), weregrown in galactose-containing media. Glucose was then addedto repress transcription of the reporter. RNA was recoveredfrom cells harvested at the indicated times after repression(min), separated on a 6% polyacrylamide gel, and detected byhybridization with an oligonucleotide probe specific for theMFA2 mRNA (oRP121) (Muhlrad et al., 1994). The lengthand mobility of size standards are indicated to the right of thegel. (B) The level of MFA2 mRNA in each lane shown in (A)was quantified by phosphor-imaging and then plotted as afunction of time. The plotted values have been normalized to anSCR1 loading control. The level of MFA2 is shown, expressedas a percentage of that found prior to the repression.


Figure 7. Decay of PGK1 in PAB1 and pab1-53 strains. (A)Yeast strains YAS2193 (PAB1) and YAS 2196 (pab1-53),carrying the GAL::PGK1 reporter (pRP602), were grown ingalactose-containing media. Glucose was then added to represstranscription of the reporter. RNA was recovered from cellsharvested at the indicated times after repression (min), separ-ated on a 1·2% agarose–formaldehyde gel, and detected byhybridization with an oligonucleotide probe specific for thePGK1 mRNA (oRP121) (Muhlrad et al., 1994). (B) The level ofPGK1 mRNA in each lane shown in (A) was quantified byphosphor-imaging and then plotted as a function of time. Theplotted values have been normalized to an SCR1 loadingcontrol. The level of PGK1 is shown, expressed as a percentageof that found prior to the repression.

analysis of the decay of newly transcribed mRNA.For this assay, the rpb1-1 allele was introducedinto the PAB1 and pab1-53 strains. rpb1-1 is a tsallele of a subunit of RNA Pol II and shifting cellscarrying this allele to 37)C leads to rapid, specificshut-off of Pol II transcription (Nonet et al., 1987).Yeast strains were treated with 0·5 m Cu for12 min to induce synthesis of CUP1 mRNA, andthe cells were then transferred to Cu-free mediumat 37)C to repress all Pol II transcription. Thedecay of the newly transcribed CUP1 mRNA wasthen assessed (Figure 8). CUP1 synthesis was

strongly induced in both strains, although, asshown earlier, the level of CUP1 mRNA is lowerin the pab1-53 strain. Shifting the cells to 37)Crepressed CUP1 transcription in both wt andmutant strains although, for reasons which are notknown, this shut-off was reproducibly more rapidin the wt strain (Figure 8). There was no delay inthe entry of CUP1 into the decay pathway, as seenby the commencement of tail shortening at com-parable time-points in the wt and mutant (4–6 mintime-points). To determine the decay rate of theCUP1 mRNA, the level of RNA was calculated ateach time-point and plotted as a function of time

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Figure 8. Transcription pulse-chase of CUP1 mRNA in PAB1and pab1-53 strains. Yeast strains YAS2205 (PAB1) (A) andYAS2206 (pab1-53) (B) were grown in minimal media tomid-log phase. Transcription of the CUP1 gene was induced bythe addition of 0·5 m CuSO4 for 12 min before transcriptionwas thermally repressed by shifting the cells to 37)C. RNA wasrecovered from cells harvested at the indicated times (min),separated on 6% polyacrylamide gels, and then detected byhybridization to CUP1 or SCR1 probes. Lane 1, 0 min time-point RNA sample treated with RNAse H and oligo(dT) toremove the poly(A) tail; lane 2, RNA from uninduced cells;lanes 3–15, RNA recovered from cells harvested at the indi-cated times following the temperature shift. (C) The level ofCUP1 in each lane of the gel shown in panel A was quantifiedby phosphor-imaging and then plotted as a function of time.The plotted values have been normalized to an SCR1 loadingcontrol. The level of CUP1 is shown, expressed as a percentageof the maximum level of CUP1 found after induction.


(Figure 8C). Averaging three separate exper-iments, the half-life of CUP1 mRNA in the wtstrain was found to be 13 min, and in the pab1-53mutant strain 21 min (Table 4).

The pattern of decay of CUP1 mRNA in the wtstrain follows the scheme previously described forother mRNAs: deadenylation of the bulk of themessage precedes decay. Newly synthesized CUP1mRNA carries long poly(A) tails (Figure 8A, lane3) and as the mRNA is deadenylated it migratesmore rapidly in the gel (Figure 8A, lanes 3–6).Most of the mRNA was shortened to an oligo-adenylated form, containing 10–12 adenylate resi-dues, before being degraded (Figure 8A, lanes5–13). Due to the use of alternative transcriptionsites (Karin et al., 1984), the oligo(A) form ofCUP1 mRNA is predominantly a doublet (bestseen at later time-points). In the mutant strain, theCUP1 decay profile is quite different. Unlike thewt strain, where distinct species representing CUP1with an oligo(A) tail are seen (Figure 8A, lanes 6,7 and 8), no oligo(A)-CUP1 is detected in thepab1-53 mutant strain (Figure 8B). In fact, thebulk of the CUP1 mRNA decays while still carry-ing a long poly(A) tail. This same effect was alsoobserved with MFA2 mRNA, where no mRNAwith short poly(A) tails was detected in the mutantstrain (Figure 6A).

Polyadenylated mRNA is decapped in pab1mutants

The pattern of mRNA decay in a strain carryingthe pab1-53 allele suggested that mRNA wasbeing decapped and then degraded while stillcarrying a polyadenylate tail. This phenomenon ofpremature decapping was previously described in astrain carrying a null allele of pab1, and wasconsidered to result in severe mRNA destabiliza-tion (Caponigro and Parker, 1995). To investigatewhether mRNA in pab1 mutants was decappedwhile still polyadenylated, reporter constructsdesigned to trap decay intermediates were intro-duced into these strains. The reporter constructsconsisted of either the MFA2 (Decker and Parker,1993) or the PGK1 (Muhlrad et al., 1995) genewith a poly(G) tract inserted near the 3* end of themRNA. Following decapping, Xrn1p-mediateddegradation of the reporter proceeds in a 5* to 3*direction but is blocked by the poly(G) tract.This leads to accumulation of a trapped decayintermediate (Decker and Parker, 1993; Muhlradet al., 1994; Muhlrad et al., 1995). Because

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Figure 9. Polyadenylated mRNA is decapped in pab1mutants. Strains carrying the indicated pab1 alleles were trans-formed with either pRP485, carrying the MFA2pG reporter, orpRP602, carrying the PGK1pG reporter. RNA recovered fromstrains grown to mid-log in YMGAL was separated on 6%polyacrylamide gels and detected by hybridization with anoligonucleotide probe specific to the pG tract of the reportermRNAs (oRP121). (A) RNA recovered from strains carryingthe MFA2 reporter. (B) RNA recovered from strains carryingthe PGK1 reporter. For each panel, odd-numbered lanes con-tain untreated RNA, even-numbered lanes contain RNAtreated with RNase H and oligo(dT) to remove the poly(A) tail(RNase H). In (A), both the full-length reporter, at the top ofthe blot, and decay intermediates, at the bottom, can be seen,whereas in (B), the full-length reporter is too large to enter thegel and only the decay intermediates are seen. The approximatelength of poly(A) tail on the decay intermediates, calculatedfrom the mobility of molecular weight markers run on each gel,are shown to the left. Approximately 10 ìg of RNA were loadedin each lane, but different exposures are shown to best illustrate
the decapping of polyadenylated mRNA phenomenon.
deadenylation precedes decapping, ordinarily thisdecay intermediate has only a short length ofadenylate residues at the 3* end (210 residues). Itwas found, however, that some of the pab1 mu-tants accumulated intermediates with longerstretches of adenylate residues at the 3* end. Thelength of poly(A) tail on the trapped MFA2 inter-mediate varied between strains: in strains carryingPAB1, pab1-55 or pab1-38, the majority of theintermediates possessed a tail of 10–12 adenylateresidues, whereas the majority of the intermediatesin strains carrying the pab1-66, pab1-52 andpab1-53 alleles had adenylate tails of 18–22, 25–30and 30–35 residues, respectively (Figure 9A). Simi-lar effects were observed with the PGK1 reporter(Figure 9B). Based on these data, we conclude thatdecapping of mRNA prior to its deadenylation isnot necessarily an indicator of mRNA instability,and that the presence of such intermediates is notlethal to yeast. Evidence that the decapping ofadenylated mRNA is not a lethal event has alsobeen reported by Belostotsky and Meagher (1996)in their studies on the expression of the Arabidop-sis PAB5 gene in yeast. We note that there was nota direct correlation between the mutants whichdecapped polyadenylated mRNA and those whichwere sensitive to elevated Cu/3-AT levels. Forexample, a strain carrying the pab1-66 allele accu-mulated intermediates with a poly(A) tail but hadno detectable sensitivity to Cu or 3-AT.

mRNA production is impaired in the pab1-53mutant

The rate of accumulation of mRNA was exam-ined in the pab1-53 mutant strain, since it exhibitedreduced mRNA levels without increased rates ofmRNA degradation. CUP1 mRNA was againused as a reporter. Yeast strains carrying the PAB1or pab1-53 allele were treated with Cu for varioustimes and the level of CUP1 mRNA assessed(Figure 10). There was a low level of CUP1mRNA, migrating as a smear because of thevariable length poly(A) tail, present in theuninduced cells (lanes 1 and 10). After 4 min oftreatment with 0·5 m Cu, newly produced CUP1mRNA was detected in both strains (lanes 3 and12). The level of CUP1 mRNA continued toincrease upon longer treatment with Cu. AlthoughCUP1 mRNA levels increased in both strains, therate of increase was considerably higher in thestrain carrying the wt PAB1 allele (Figure 10B). Atthe later time-points, the level of CUP1 mRNA is

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Figure 10. Kinetics of induction of CUP1 mRNA. (A) Mid-log yeast cultures of YAS2205 (PAB1) and YAS2206 (pab1-53)at 25)C were made 0·5 m CuSO4. Cells were harvested at theindicated times (min), and RNA was isolated, separated on a6% polyacrylamide gel and detected by hybridization to CUP1or SCR1 probes. (B) CUP1 levels in each lane were calculatedby phosphor-imaging, normalized for loading variation usingSCR1 levels, and plotted as a function of time. The y-axisindicates the intensity, in counts, for each band recorded by thephosphor-imager.

a function of both the rate of synthesis and the rateof decay. The levels at the early time-points, how-ever, primarily reflect the rates of production, sinceCUP1 mRNA has a half-life of 13 and 20 min inthese strains (Table 4). The differences in the ratesof accumulation of CUP1 mRNA are not readilyexplained by differences in growth rates, since aslow-growing strain carrying a cdc33 allele did notdisplay this phenotype (data not shown). Theslower initial rate of accumulation of CUP1mRNA in the pab1-53 mutant suggests that thisstrain is impaired in the production of mRNA.

DISCUSSION

We report here that certain yeast pab1 mutantsexhibit an increased sensitivity to copper and3-aminotriazole which is alleviated by overexpres-


sion of CUP1 or HIS3, respectively. Although thepab1 mutants retain the ability to sense andrespond to these substances, they show reducedaccumulation of mRNA. This decreased accumu-lation is not due to increases in mRNA degrada-tion rates, even though some of the mutants dodecap significant amounts of polyadenylatedmRNA. Instead, the lower levels appear to resultfrom inefficient production of mRNA. These datasupport the conclusion that pab1 mutants canaffect some aspect of mRNA production in theyeast cell.

Given the proposed role of Pab1p in protectingmRNA from degradation in the cytoplasm(Caponigro and Parker, 1995), it was expectedthat the low level of mRNA relative to RNAtranscribed by RNA Pol I or RNA Pol III wouldbe explained by an increased rate of mRNA turn-over in the mutant strains. In fact, this turned outnot to be the case and mRNA is more stable in thepab1-53 mutant (Table 4). Although mRNA wasnot less stable, an altered pattern of degradationwas evident, with the bulk of the mRNA beingdegraded before deadenylation was completed.This altered pattern was suggested by the lack ofappearance of MFA2 mRNA with short poly(A)tails following transcription shut-off (Figure 6),and was confirmed by following the decay ofCUP1 mRNA synthesized during a short tran-scription pulse (Figure 8). Using previously char-acterized reporters, it was found that this was ageneral phenomenon in other pab1 mutants:decapping and subsequent 5*–3* degradation wereuncoupled from deadenylation (Figure 9).

The precise nature of the link between dead-enylation and decapping remains uncertain. It isclear that there is a temporal correlation betweenthese two events, with deadenylation ordinarilypreceding decapping (Decker and Parker, 1993;Caponigro and Parker, 1996). In fact, the stabili-zation of the poly(A) tails and the partial stabili-zation of mRNAs in the pab1-53 strain providefurther correlative data. Nevertheless, our datashow that even in the absence of extensive dead-enylation, decapping and degradation of mRNAoccurs at a rate only two-fold different from nor-mal. This suggests that a major rate-determinantof decapping and mRNA degradation is not therate at which poly(A) tails are removed. Onemodel of mRNA decay has been that normal ratesof degradation require deadenylation to removePab1p, a postulated inhibitor of decapping, fromthe mRNA. Combined with our finding that a

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deletion of XRN1 does not suppress a deletion ofPAB1, these data indicate that regulation ofmRNA decay is more complex than that modelsuggests.

It was interesting that some mutant alleles ofpab1 were unable to support growth in a strainlacking the Xrn1p exonuclease. The previous find-ing that uncapped mRNA is stabilized by adeletion of XRN1 (Hsu and Stevens, 1993) pro-vides an explanation for this synthetic lethality.Ordinarily, a strain deleted for XRN1 accumulatesuncapped, unadenylated mRNA. Since this RNAcarries neither the 5* nor the 3* translation deter-minant, it would not be expected to be a substratefor the cellular translation machinery. In the pab1xrn1 double mutants, however, the RNA which ispredicted to accumulate would be uncapped andpolyadenylated. This RNA could be toxic for anumber of reasons: e.g. the presence of the poly(A)tail may drive aberrant internal initiation of trans-lation, leading to synthesis of truncated proteins;alternatively, the uncapped polyadenylated RNAcould sequester translation initiation factors with-out being translationally active itself, therebyimpairing the ability of the cell to carry out normaltranslation.

The steady-state level of an mRNA reflects thedifference between its rates of production anddegradation. Since the mRNA levels are lower inthe pab1-53 strain but mRNA stability is notdecreased, it is not surprising that the rates ofaccumulation were found to be reduced (Figure10). Furthermore, the poly(A) tail on newly tran-scribed mRNA was slightly longer in the mutantpab1 strain (Figure 10, cf. lanes 8 and 9 with lanes17 and 18), a phenotype previously reported in apab1 null strain (Caponigro and Parker, 1995).These data indicate that normal production ofmRNA is impaired in pab1 mutants. Our data donot address the specific role of Pab1p in theproduction of mRNA, but work from other lab-oratories has proposed that Pab1p is involved inthe cleavage and polyadenylation reactions takingplace at the 3*-end of mRNA in the nucleus(Amrani et al., 1997; Minvielle-Sebastia et al.,1997). In that regard, it is interesting that mutantsin other cleavage and polyadenylation factors alsolead to a decrease in the levels of mRNA: aphenotype similar to that seen in the pab1-53mutant (Minvielle-Sebastia et al., 1991; Birse et al.,1998). The involvement of Pab1p in the diverseprocesses of polyadenylation, translation andmRNA turnover make the in vivo analysis of


mutants difficult; however, the availability ofin vitro system should facilitate an understandingof the roles of Pab1p in the cell.

ACKNOWLEDGEMENTS

We thank members of our laboratory for adviceduring the course of this work and for criticalreading of the manuscript. We thank R. Parker, A.Hinnebusch, A. Johnson, R. Young and S. Peltzfor plasmids and strains used in this study. Thiswork was supported by Grants to A.B.S. from theAmerican Cancer Society (No. 82666), theNational Institutes of Health (No. R01-GM50308), and the Hellman Family Fund.

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Decapping of stabilized, polyadenylated mRNA in yeastpab1 mutants