Mechanism of mRNA deadenylation: evidence for a molecular...

Mechanism of mRNA deadenylation:evidence for a molecular interplaybetween translation termination factoreRF3 and mRNA deadenylasesYuji Funakoshi,1,2,6 Yusuke Doi,1,6 Nao Hosoda,1,6 Naoyuki Uchida,3,6 Masanori Osawa,4

Ichio Shimada,4 Masafumi Tsujimoto,2 Tsutomu Suzuki,5 Toshiaki Katada,3 andShin-ichi Hoshino1,7

1Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya467-8603, Japan; 2Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama 351-0198, Japan; 3Department ofPhysiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan;4Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033,Japan; 5Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo113-0033, Japan

In eukaryotes, shortening of the 3�-poly(A) tail is the rate-limiting step in the degradation of most mRNAs,and two major mRNA deadenylase complexes—Caf1–Ccr4 and Pan2–Pan3—play central roles in this process,referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive.Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay ofmRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNAdeadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1–Ccr4 and Pan2–Pan3.Interestingly, translation termination complexes eRF1–eRF3, Pan2–Pan3, and Caf1–Ccr4 competitivelyinteract with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively,mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized fortheir contacts. A translation-dependent exchange of eRF1–eRF3 for the deadenylase occurs on PABPC1.Consequently, PABPC1 binding leads to the activation of Pan2–Pan3 and Caf1–Ccr4. From these results, wesuggest a mechanism of mRNA deadenylation by Pan2–Pan3 and Caf1–Ccr4 in cooperation with eRF3 andPABPC1.

[Keywords: Translation termination; deadenylation; eRF3; PABPC1]

Supplemental material is available at http://www.genesdev.org.

Received July 26, 2007; revised version accepted October 11, 2007.

Control of mRNA decay is a fundamentally importantstep in determining the amount of protein producedfrom the mRNA by translation, and mRNA decay is in-timately linked to and regulated by translation. In eu-karyotes, decay of most mRNAs is initiated by shorten-ing of the poly(A)-tail at the 3� end (Shyu et al. 1991;Muhlrad and Parker 1992), which is followed by two gen-eral pathways: the 5�-to-3� and 3�-to-5� exonucleolyticdegradation pathways. The poly(A) tail-shortening pro-cess, referred to as deadenylation, is the first and rate-limiting step, and is also the most efficient step in con-trolling mRNA decay (Decker and Parker 1993).

Two distinct enzyme complexes involved in the de-adenylation process have been identified in yeast(Daugeron et al. 2001; Tucker et al. 2001) and mammals(Yamashita et al. 2005). One major cytoplasmic dead-enylase complex is Ccr4–Caf1, which was originally im-plicated in the control of transcription (Denis and Chen2003). As suggested by the significant homology withknown 3�-to-5� exonucleases, both Ccr4 and Caf1 havecatalytic activity of the deadenylase (Chen et al. 2002;Tucker et al. 2002; Thore et al. 2003; Viswanathan et al.2004; Bianchin et al. 2005). Another major deadenylasecomplex, PAN, was identified in yeast (Sachs and Dear-dorff 1992) and in mammal (Uchida et al. 2004). PAN isa heterodimeric protein consisting of catalytic Pan2 andregulatory Pan3 subunits (Brown et al. 1996; Uchida etal. 2004). In contrast to Ccr4–Caf1, PAN is characterizedby the requirement of PABPC1 for its deadenylating ac-tivity: Pan2 is activated by binding to PABPC1 through

6These authors contributed equally to this work.7Corresponding author.E-MAIL [email protected]; FAX 81-52-836-3427.Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1597707.

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the interaction with Pan3 (Mangus et al. 2004; Uchidaet al. 2004).

Since deadenylation is the rate-limiting step formRNA decay, the enzymatic activities of the twomRNA deadenylases constitute a major target for thecontrol of mRNA decay. Recent findings demonstratedthat selective recruitment of deadenylating enzymes tothe substrate mRNA is involved in the decay mecha-nism for specific mRNAs (Semotok et al. 2005; Finouxand Seraphin 2006; Goldstrohm et al. 2006). However,the general mechanism triggering deadenylation ofmRNA has not been elucidated. Several lines of evidencehave revealed a direct link between translation termina-tion and mRNA decay.

In eukaryotes, translation termination is governed byheterodimeric releasing factors eRF1 and eRF3 (Frolovaet al. 1994; Stansfield et al. 1995; Zhouravleva et al.1995). eRF1 directly recognizes all three stop codons inthe ribosomal A site to release the completed polypep-tide chain from the ribosome (Frolova et al. 1994), andeRF3 stimulates the termination reaction in a GTP-de-pendent manner (Zhouravleva et al. 1995). The eRF3gene is conserved from yeast to mammals. In the yeastSaccharomyces cerevisiae, eRF3 is encoded by the essen-tial gene SUP35 (Stansfield et al. 1995), and the mam-malian eRF3 gene GSPT was first identified by its abilityto complement the temperature-sensitive growth arrestphenotype of a sup35/gst1-1 mutant S. cerevisiae that isdefective in the G1-to-S-phase transition (Hoshino et al.1989). Later, two subtypes of GSPT genes, GSPT1 andGSPT2, were identified (Hoshino et al. 1998). eRF3 con-sists of two domains: the unique N-terminal region (Ndomain) and the C-terminal region (C domain), whichcontains eEF1A-like GTP-binding motifs. The C domainof eRF3 is sufficient to fulfill the functions of eRF3 intermination (Zhouravleva et al. 1995).

In addition to translation termination, eRF3 functionsin translation termination-coupled events. We showedpreviously that eRF3 interacts with poly(A)-binding pro-tein (PABPC1) through its N domain (Hoshino et al.1999). The interaction is evolutionarily conserved fromyeast to mammals (Hoshino et al. 1999; Kozlov et al.2001; Cosson et al. 2002; Uchida et al. 2002; Hosoda etal. 2003). In yeast, eRF3 regulates the initiation of nor-mal mRNA decay at the poly(A) tail-shortening stepthrough the interaction with PABPC1 in a mannercoupled to translation termination (Hosoda et al. 2003).Recently, Tpa1—which interacts with eRF1/eRF3 andaffects translation termination, deadenylation, andmRNA decay—was identified (Keeling et al. 2006). Onthe other hand, in nonsense codon-containing mRNA,translation termination is thought to occur via the ter-mination complex eRF1/eRF3 at the premature termina-tion codon. eRF1/eRF3 associates with Upf1/Upf2/Upf3to form a “surveillance complex” and triggers rapidmRNA decay via the nonsense-mediated mRNA decay(NMD) pathway (Wang et al. 2001; Kobayashi et al. 2004;Kashima et al. 2006).

In this study, we examined the mechanism of mRNAdeadenylation and obtained evidence for a molecular in-

terplay between translation termination factor eRF3 andmRNA deadenylases. We demonstrate that eRF3-medi-ated deadenylation is catalyzed by both of the two majormRNA deadenylase complexes in yeast and humans. In-terestingly, translation termination complex eRF1–eRF3and the two mRNA deadenylase complexes competi-tively bind to the PABC domain of PABPC1. The PAM2motifs found in both eRF3 and the two deadenylase com-plexes are responsible for their binding to PABPC1 andmRNA deadenylation. The termination complex and thedeadenylase complex are exchanged on PABPC1 in atranslation-dependent manner. Recruitment of the twodeadenylase complexes to PABPC1 leads to the activa-tion of both enzymes. These results provide a molecularbasis for the mechanism of mRNA deadenylation.

Results

The two major mRNA deadenylases, Pan2–Pan3and Caf1–Ccr4, are involved in the eRF3-mediateddeadenylation of mRNA in S. cerevisiae

Using S. cerevisiae as a model organism, we showed pre-viously that eRF3 mediates deadenylation and decay ofnormal mRNA in a manner coupled to translation ter-mination (Hosoda et al. 2003). To identify the deadenyl-ases involved in the eRF3-mediated deadenylation, wefirst examined the possible involvement of the twomajor mRNA deadenylases, Pan2–Pan3 and Caf1–Ccr4,by comparing phenotypes in strains pan2� and ccr4�,and a strain in which the N-terminal domain of eRF3(SUP35N) was overexpressed. In this study, we used thestrain overexpressing SUP35N, since this strain and theeRF3 mutant strains sup35�N and sup35ts show thesame phenotypes regarding mRNA deadenylation anddecay (Hosoda et al. 2003). We first analyzed the poly(A)tail length of total steady-state mRNA in vivo. Afterlabeling with [32P]pCp, total RNA was digested withRNaseA, and the remaining intact poly(A) tails were re-solved on polyacrylamide gels (Fig. 1A,B). As reportedpreviously, the overexpression of SUP35N led to an in-crease in bulk mRNA poly(A) tail lengths (Hosoda et al.2003). Similarly, a deletion in PAN2 also resulted in anincrease in the average poly(A) tail length. PhosphorImageranalysis showed that the average length increased ∼20nucleotides (nt) in both strains compared with the wild-type strain. The changes in poly(A) tail length on mRNAin pan2� overexpressing SUP35N were comparable withthose found in either pan2� or the SUP35N-overexpress-ing strain. Thus, the deadenylation defect in theSUP35N-overexpressing strain was not exacerbated bypan2�. These results suggest that Pan2 is involved atleast in part in eRF3-mediated deadenylation.

However, a previous study reported that pan2� doesnot show any defect in the rate of mRNA deadenylationor decay (Tucker et al. 2001). Therefore, we next exam-ined the decay of an MFA2pG reporter transcript thatwas under the control of a tetracycline-regulatable pro-moter system. In this system, the reporter gene is spe-cifically and rapidly suppressed in the presence of doxy-

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cycline. The decay rate of the mRNA was measured aftertranscriptional shutoff by the addition of doxycycline.Consistent with previous reports, pan2� did not showany apparent defect in the decay of the MFA2pG mRNA(t1/2 = 15 min, cf. wild type and pan2�) (Fig. 1C). In ad-dition, the poly(A) tail of MFA2pG examined using high-resolution polyacrylamide Northern blot shortened at arate of 2.0 nt per minute, which was comparable withthe rate in wild-type cells (Fig. 1D). In sharp contrast,both the decay and the poly(A) tail-shortening rates ofthe MFA2pG mRNA were substantially reduced in thestrain expressing SUP35N (t1/2 = 46 min and 0.45 nt perminute), although the distribution of the poly(A) taillength at time 0 was comparable with that of pan2�.Furthermore, overexpression of SUP35N reduced therates of both the decay and the poly(A) tail shortening ofthe mRNA in the pan2� strain. These results suggestthat additional deadenylases are involved in eRF3-medi-ated deadenylation and decay. In view of the defect in thedeadenylation rates, Ccr4 is one such candidate. As re-ported previously, both the decay and the poly(A) tailshortening of the MFA2pG mRNA were substantially

reduced in the strain ccr4� (t1/2 = 68 min and 0.35 nt perminute) (Tucker et al. 2001), which is similar to the phe-notype of the SUP35N-overexpressing strain. In addi-tion, SUP35N had no significant effect on the rates ofboth deadenylation and decay of the mRNA in the ccr4�strain but led to an increase in the poly(A) tail length ofeither MFA2 mRNA or bulk mRNA in the ccr4� strainat steady state (Fig. 1, cf. D and A). Thus, the phenotypeof the longer poly(A) tail of steady-state mRNA in theSUP35N-overexpressing strain is explained by the defectin Pan2, and the phenotype of the reduced rates of bothdeadenylation and decay is explained by the defect inCcr4. Taken together, these results indicate that two majormRNA deadenylases, Pan2–Pan3 and Caf1–Ccr4, are in-volved in the eRF3-mediated deadenylation of mRNA.

The two major mRNA deadenylases, hPan2–hPan3and hCaf1–hCcr4, are involved in the eRF3-mediateddeadenylation of mRNA in mammals

Next, we examined whether hPan2–hPan3 and hCaf1–hCcr4 are also involved in the eRF3-mediated mRNA

Figure 1. Comparison of the length of poly(A)tails and decay rates in S. cerevisiae. (A) pKT10or pKT10-SUP35N, which overexpresses the Ndomain of eRF3 (SUP35N) under the control ofGAL1 UAS, was introduced into wild-type,pan2�, and ccr4� strains. Total RNAs at thesteady-state level from the strains were 3�-la-beled with [32P]pCp and digested with RNaseA.The remaining mRNA poly(A) tails were sepa-rated on a 12% polyacrylamide–7 M urea gel andvisualized by autoradiography. (B) The resultingautoradiograph from A was analyzed by densito-metric tracing. (C) p9L-MFA2pG was introducedinto the same strains as used in A. After transcrip-tional repression by adding doxycycline, the cellswere collected at the indicated time points. (Left)Total RNAs were separated on a 1% formamide-agarose gel, and MFA2pG mRNA was detectedby Northern blotting. (Right) The amount ofMFA2pG mRNAs at each time point was mea-sured. The half-lives (t1/2) of the mRNAs werederived from three independent experiments andare shown as the average and the standard errorof the mean (SEM) in the left panel. (D) The dead-enylation rates of MFA2pG mRNA poly(A) tailsin the strains shown in C were analyzed by apolyacrylamide Northern blot analysis. (Left)The RNA samples prepared in C were separatedon a 4% polyacrylamide–8 M urea gel. The RNAsamples from 0-min time points were treatedwith RNaseH and oligo(dT) to indicate the posi-tion of the deadenylated mRNA. (Right) Thesizes of the longest poly(A) tails were measured.

Mechanism of mRNA deadenylation

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deadenylation in mammalian cells. We monitoredmRNA deadenylation and decay kinetics by using a tet-racycline regulatory transcriptional pulse-chase ap-proach in HeLa cells. In this experiment, �-globinmRNA was used as a reporter, since the transcript is wellcharacterized and therefore provides an ideal model sys-tem to study mRNA stability. T-Rex-HeLa cells weretransfected with the �-globin gene, transcription of thegene was driven by tetracycline for a short period oftime, and the metabolism of newly transcribed mRNAswas followed over time. As reported previously, �-globinmRNA showed slow and biphasic decay kinetics with ahalf-life of ∼12 h (Fig. 2A, left panel). A slow and syn-chronous deadenylation occurred in the first phase (0–3h), which was followed by a fast and less-synchronousdeadenylation with a concomitant decay of the mRNAbody (second phase) (Supplementary Fig. 1C, left panel).Overexpression of wild-type hPan2 increased the rate ofdeadenylation in the first phase (0–3 h) (Fig. 2A, lanes6,7), whereas a comparable amount of hPan2 mutantthat had no deadenylase activity (Uchida et al. 2004) re-duced the rate (Fig. 2A, lanes 11,12). On the other hand,overexpression of wild-type hCaf1 increased the rate of

deadenylation (Fig. 2B; Supplementary Fig. 1) and en-hanced the decay of the mRNA body in the second phase(Fig. 2B, lane 7; Supplementary Fig. 1C). In sharp con-trast to this, a nuclease-deficient hCaf1 mutant in whicha catalytically essential aspartate residue was mutatedto alanine drastically reduced the rate of deadenylationand decay of mRNA body in the second phase and sta-bilized the mRNA (Fig. 2B, lanes 13–15; SupplementaryFig. 1C). The expression of hPan2 or hCaf1 protein wasconfirmed by Western blot (Fig. 2D,E). These results con-firm the previous results showing that hPan2–hPan3 andhCaf1–hCcr4 function in the first and second phases ofmRNA deadenylation, respectively (Yamashita et al.2005).

We then examined whether eRF3 mediates deadenyla-tion of mRNA in HeLa cells. As shown in Figure 2C,ectopic expression of an N-terminal domain of eRF3(eRF3N) reduced the rate of deadenylation in both thefirst and the second phases. The expression of the eRF3Nwas confirmed by Western blot (Fig. 2F). Collectively,these results suggest that eRF3 mediates deadenylationthrough hPan2–hPan3 and hCaf1–hCcr4 in mammaliancells.

Figure 2. Comparison of deadenylation rates in HeLacells. (A) T-Rex-HeLa cells were transfected withpFlag-CMV5/TO-G1 reporter and either pFlag-CMV5(lanes 1–5), pFlag-hPan2 (lanes 6–10), or pFlag-hPan2D1085A (lanes 11–15). One day later, �-globin mRNAwas induced by tetracycline for 2.5 h, and then cellswere harvested at the specified time after �-globinmRNA transcription shutoff. (Left) �-Globin mRNAwas detected by Northern blot analysis. (Lane 16) Todetermine the overall poly(A) tail length and the dead-enylation endpoint (arrow) of �-globin mRNA, �-glo-bin mRNAs at the steady-state level (24-h induction)are shown. (Right) The average length of poly(A) tailsat each time point was measured. (B) As in A, exceptthat cells were transfected with pHA-CMV5 (lanes1–5), pHA-hCaf1 (lanes 6–10), or pHA-hCaf1 D161A(lanes 11–15), and transcriptional pulse-chase analysiswas performed. (C) As in A, except that cells weretransfected with pCMV-Myc (lanes 1–5) or pCMV-Myc-eRF3 N (lanes 6–10), and transcriptional pulse-chase analysis was performed. (D–F) Total cell lysateswere analyzed by immunoblotting using anti-Flag (D,top), anti-HA (E, top), anti-Myc (F, top), or anti-GAPDH (D–F, bottom).

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hPan3 and eRF3 competitively bind to PABPC1

In mammals, we showed that eRF3 as well as hPan3interacts with PABPC1 (Hoshino et al. 1999; Uchida etal. 2002, 2004). The present results showing that eRF3-mediated deadenylation is catalyzed in part by hPan2–hPan3 (Fig. 2) led us to speculate that, in mammaliancells, eRF3 mediates mRNA deadenylation by hPan2–hPan3 through interaction with PABPC1. Therefore, weinvestigated the interactions among eRF3, hPan2–hPan3, and PABPC1. To this end, we first examinedwhether eRF3 and hPan3 could simultaneously interactwith PABPC1. When an extract of COS-7 cells express-ing Flag-eRF3 and HA-hPan3 was immunoprecipitatedwith anti-Flag antibody, the precipitated fraction con-tained endogenous PABPC1 but not HA-hPan3 (Fig. 3A,lane 4). On the other hand, anti-Flag immunoprecipitatefrom extracts of cells expressing Flag-hPan3 contained

PABPC1 but not eRF3 (data not shown). These resultssuggest that both hPan3 and eRF3 bind with PABPC1,but each protein is part of a distinct complex, and eRF3might compete with hPan3 for PABPC1 binding. To testthis idea, we examined whether hPan3 and eRF3 interactwith the same region of PABPC1. Flag-PABPC1 or Flag-PABPC1�C lacking the eRF3-binding domain was coex-pressed with HA-hPan3, and an immunoprecipitation as-say using anti-Flag antibody was performed. As shown inFigure 3B, Flag-PABPC1, but not Flag-PABPC1�C wascapable of coprecipitating with eRF3, which is consis-tent with our previous study using a yeast two-hybridsystem (Hoshino et al. 1999). A similar result was alsoobtained for the coprecipitation of HA-hPan3 (Fig. 3B,top). Thus, both eRF3 and hPan3 require the C-terminaldomain of PABPC1 for their binding.

Next, we examined whether hPan3 actually competeswith eRF3 for PABPC1 binding. A complex consisting of

Figure 3. hPan3 and eRF3 competitively bind toPABC domain of PABPC1 via their PAM2 motifs.(A) COS-7 cells were transfected with pHA-hPan3 and either pFlag-CMV5 (lanes 1,3) orpFlag-eRF3 (lanes 2,4). The cell extracts weresubjected to an immunoprecipitation assay (IP)using anti-Flag antibody. The immunoprecipi-tates (lanes 3,4) and the inputs (lanes 1,2, 20% ofthe amount immunoprecipitated) were analyzedby immunoblotting using the indicated antibod-ies. (B) COS-7 cells were transfected with pHA-hPan3 and either pFlag-CMV5 (lanes 1,4), pFlag-PABPC1 (lanes 2,5), or pFlag-PABPC1�C (lanes3,6). The cell extracts were subjected to an im-munoprecipitation assay and immunoblot analy-ses as described in A. (C) COS-7 cells were trans-fected with pHA-hPan2 and either pFlag-CMV5(lane 1) or pFlag-hPan3 (lanes 2–4), and the cellextracts were subjected to an immunoprecipita-tion assay using anti-Flag antibody. Coprecipi-tated proteins were incubated with buffer alone(lane 2), recombinant GST-eRF3 (58–204)-(His)6(lane 3), or GST-eRF3 (80–204)-(His)6 (lane 4).The remaining proteins were separated by SDS-PAGE, and immunoblot analysis was carried outwith the indicated antibodies. (D) COS-7 cellswere transfected with pHA-hPan3 and eitherpFlag-CMV5 (lanes 1,6), pFlag-PABPC1 (lanes2,7), pFlag-PABPC1 F567A (lanes 3,8), pFlag-PABPC1 K580A/L585A (lanes 4,9), or pFlag-PABPC1 F567A/K580A/L585A (lanes 5,10). Thecell extracts were subjected to an immunopre-cipitation assay and immunoblot analysis as de-scribed in A. (Top) PABPC1 protein is schemati-cally represented. Gray bar indicates PABC do-main. The partial sequence of PABC is alsoshown. The amino acid residues that were mu-tated in the experiment are shaded. (E) COS-7

cells were transfected with either pHA-CMV5 (lanes 1,6), pHA-hPan3 (lanes 2,7), pHA-hPan3 Y57A (lanes 3,8), pHA-hPan3 F93A (lanes4,9), or pHA-hPan3 Y57A/F93A (lanes 5,10). The cell extracts were subjected to an immunoprecipitation assay using anti-HA antibody.The immunoprecipitates (lanes 6–10) and the inputs (lanes 1–5, 10% of the amount immunoprecipitated) were analyzed by immu-noblotting using the indicated antibodies. (Top) hPan3 protein is schematically represented. Gray bars indicate putative PAM2 motifs.The sequences of the two putative PAM2 motifs are also shown. The amino acid residues that were mutated in the experiment areshaded.





Flag-hPan3, HA-hPan2, and PABPC1 was prepared fromCOS-7 cells expressing both Flag-hPan3 and HA-hPan2by means of immunoprecipitation with anti-Flag anti-body. Then the complex was incubated with recombi-nant eRF3 (58–204) (a fragment encompassing amino ac-ids 58–204) or eRF3 (80–204), which contained thePABPC1-binding region or not, respectively (Uchida etal. 2002). As shown in Figure 3C, eRF3 (58–204) but noteRF3 (80–204) effectively inhibited PABPC1 binding toFlag-hPan3, while the interaction between HA-hPan2and Flag-hPan3 was not affected. Thus, we conclude thathPan3 competes with eRF3 for PABPC1 binding.

The PAM2 motif of hPan3 and PABC domainof PABPC1 are responsible for the interactionbetween hPan3 and PABPC1

We reported previously that a 22-amino-acid region atthe N terminus of eRF3 is critical for the binding to theC-terminal domain of PABPC1 (PABC) (Uchida et al.2002). The PABC domain has been characterized as aprotein–protein interaction domain that recruits varioustranslation or mRNA-processing factors possessing thePABPC1-interacting motif (PAM2) to the mRNA poly(A)tail (Kozlov et al. 2001). The PAM2 motif is also found inthe N-terminal 22-amino-acid region of eRF3 (Uchida etal. 2002). Thus, eRF3 binds to PABC through its PAM2motif. hPan3 also binds to the C-terminal domain ofPABPC1 competitively with eRF3. These results led usto speculate that hPan3 competes with eRF3 by bindingto the PABC domain of PABPC1. As expected, we iden-tified two putative PAM2 motifs at the N-terminal re-gion of hPan3 (Fig. 3E). We therefore tested the idea bysite-directed mutagenesis of the PABC domain and im-munoprecipitation with extracts from COS-7 cells ex-pressing the mutants. As shown in Figure 3D, the bind-ing of hPan3 with PABPC1 was abolished by mutation ofPhe567 to alanine (F567A) (lane 8), and also by a double(K580A/L585A) (lane 9) and a triple (F567A/K580A/L585A) (lane 10) mutation in the PABC domain ofPABPC1. The same results were obtained for eRF3 (Fig.3D, top panel). These results indicate that hPan3 as wellas eRF3 binds to PABC. Next, we examined whetherhPan3 binds to PABC domain through its PAM2 motifs.As shown in Figure 3E, the binding of PABPC1 was notaffected by mutation of Tyr57 to alanine (Y57A) (lane 8)in the first PAM2 motif of hPan3, but was almost com-pletely abolished by F93A mutation in the second PAM2motif (lane 9) and also by their double mutation (Y57A/F93A) (lane 10). Thus, hPan3 binds to PABPC1 with thesecond PAM2 motif. From these results, we concludethat hPan3 and eRF3 competitively bind to the PABCdomain of PABPC1 through their PAM2 motifs.

Caf1–Ccr4 interacts with PABPC1 through Tobby using PAM2–PABC contact

In a previous study, we demonstrated that PABPC1 isrequired for the activation of the deadenylating activity

of hPan2–hPan3 in vitro (Uchida et al. 2004). However,the role of PABPC1 in the activity of the hCaf1–hCcr4complex is still controversial; in yeast, Pab1 is inhibitoryto Ccr4 in vitro (Tucker et al. 2002), whereas a pab1�strain severely blocks Ccr4-catalyzed deadenylation invivo (Caponigro and Parker 1995). To address whetherPABPC1 is also involved in the activation of the hCaf1–hCcr4 complex, we first examined the physical interac-tion between hCaf1 and PABPC1 by an immunoprecipi-tation experiment. HeLa cells expressing Flag-PABPC1and HA-Caf1 were immunoprecipitated using anti-HAantibody. Flag-PABPC1 was capable of coprecipitatingwith HA-hCaf1 (Fig. 4A, lane 6). RNaseA treatment didnot abolish the binding, which indicates that no RNAwas required for the binding (Fig. 4A, lane 8). Moreover,the binding is indirect because a GST pull-down experi-ment using purified recombinant PABPC1 and GST-hCaf1 showed no interaction (Fig. 4B, lane 1). We there-fore searched for a factor that mediates hCaf1–PABPC1binding. Previous findings independently demonstratedthat Tob binds to both PABPC1 (Okochi et al. 2005) andhCaf1 (Ikematsu et al. 1999). This led us to speculatethat Tob mediates hCaf1–PABPC1 binding. As expected,purified recombinant PABPC1 bound to GST-hCaf1 inthe presence of Tob (Fig. 4B). To further confirm that Tobacts as a bridge between hCaf1 and PABPC1, we exam-ined the structural requirements for the interaction. Ei-ther Flag-tagged Tob or its deletion mutants were ex-pressed in COS-7 cells, and immunoprecipitation experi-ments using anti-Flag antibody were performed. Flag-Tob coprecipitated with both PABPC1 and HA-Caf1 (Fig.4C, lane 6). On the other hand, truncation of the Tob Cterminus (127 amino acids) led to a loss of interactionwith PABPC1 but not with HA-hCaf1. Conversely, N-terminal truncation of Tob (109 amino acids) led to a lossof interaction with HA-hCaf1 but not with PABPC1.Taken together, these results indicate that PABPC1 andhCaf1 bind to the C- and N-terminal region of Tob, re-spectively, to form a complex.

The above results demonstrate that a PAM2 motif ofhPan3 and the PABC domain of PABPC1 play essentialroles for their binding. We therefore asked whether thecombination of PAM2 and PABC is also involved in theinteraction between Tob and PABPC1 first by searchingfor PAM2 motifs in the sequence of Tob. As expected, weidentified two putative PAM2 motifs in the C-terminalregion of Tob (Fig. 4D). Recently, PAM2 motifs in Tob2,a distinct subtype of Tob, have been reported (Lim et al.2006). We therefore approached the above question bysite-directed mutagenesis and immunoprecipitationwith extracts from COS-7 cells expressing the mutants.As shown in Figure 4D, mutation of Phe139 to alanine(F139A) in the first PAM2 motif had no effect on thebinding of PABPC1, whereas mutation of Phe274 to ala-nine (F274A) in the second PAM2 motif completely abol-ished the binding. These results indicate that PABPC1binds to the second PAM2 motif of Tob. Next we exam-ined whether Tob binds to the PABC domain of PABPC1.As shown in Figure 4E, the binding of Tob was com-pletely abolished by mutation of Phe567 to alanine

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(F567A) (lane 8), and also by a double (K580A/L585A)(lane 9) and a triple (F567A/K580A/L585A) mutation(lane 10) in PABPC1. Thus, we conclude that Tob bindsto the PABC domain of PABPC1 through a PAM2 motifand bridges between PABPC1 and Caf1 to form a com-plex.

A translation-dependent exchange of eRF3for hPan2–hPan3 occurs on PABPC1 in living cells

Based on the structural evidence described above, wenext examined the possibility that competition amongeRF3, hCaf1–hCcr4, and hPan2–hPan3 occurs onPABPC1 in living cells. First, Flag-hPan3 and GST-eRF3(1–204) were expressed in COS-7 cells, and immuno-precipitation experiments were performed with anti-Flag antibody. The amount of PABPC1 coprecipi-tated with Flag-hPan3 was decreased by increasing theamount of GST-eRF3 expressed in cells (Fig. 5A, top).Similar results were obtained with Tob; the amount of

PABPC1 coprecipitated with Flag-Tob was inverselycorrelated with the amount of eRF3-(His)6 expressedin cells (Fig. 5B, top). These results suggest the possibil-ity that hPan2–hPan3 and hCaf1–hCcr4 each com-petes with eRF3 for the binding with PABPC1 in livingcells.

Since hPan2–hPan3 and hCaf1–hCcr4 are involved ineRF3-mediated mRNA deadenylation, which is coupledto translation termination, we expected that a change oftranslation status might affect interactions of eRF3,hPan3, and Tob with PABPC1. Therefore, we examinedthe effect of a translation inhibitor, cycloheximide(CHX), on the interactions. Cells expressing Flag-eRF3were treated with CHX and subjected to immunoprecipi-tation using anti-Flag antibody. Figure 5C shows that theamounts of Flag-eRF3 and eRF1 precipitated were almostthe same in the presence and absence of CHX, whileCHX treatment markedly increased the amount of co-precipitated PABPC1. In contrast, a comparative experi-ment conducted using cells expressing Flag-hPan3 and

Figure 4. hCaf1 binds to PABC domain ofPABPC1 via Tob with PAM2 motifs. (A) HeLacells were transfected with pFlag-PABPC1 and ei-ther pHA-CMV5 (lanes 1,3,5,7) or pHA-hCaf1(lanes 2,4,6,8). The cell extracts were subjected toan immunoprecipitation assay using anti-HA an-tibody in the presence or absence of RNaseA.The immunoprecipitates (lanes 5–8) and the in-puts (lanes 1–4, 10% of the amount immunopre-cipitated) were analyzed by immunoblotting us-ing the indicated antibodies. (B) RecombinantPABPC1 and GST-hCaf1 prepared from E. coliwere subjected to a GST pull-down assay in thepresence (lane 2) or absence (lane 1) of recombi-nant Tob (1–285). Immunoblot analyses wereperformed with the indicated antibodies. (C)COS-7 cells were transfected with pHA-hCaf1and either pMEFlag (lanes 1,5), pMEFlag-Tob(lanes 2,6), pMEFlag-Tob (1–218) (lanes 3,7), orpMEFlag-Tob (110–345) (lanes 4,8). The cell ex-tracts were subjected to an immunoprecipitationassay using anti-Flag antibody. The immunopre-cipitates (lanes 5–8) and the inputs (lanes 1–4,20% of the amount immunoprecipitated) wereanalyzed by immunoblotting using the indi-cated antibodies. (D) COS-7 cells were trans-fected with pHA-hCaf1 and either pMEFlag(lanes 1,6), pMEFlag-Tob (lanes 2,7), pMEFlag-Tob F139A (lanes 3,8), pMEFlag-Tob F274A(lanes 4,9), or pMEFlag-Tob F139A/F274A (lanes5,10). The cell extracts were subjected to an immu-noprecipitation assay and immunoblot analyses asdescribed in C. (Top) Tob protein is schematicallyrepresented. Gray bars indicate putative PAM2motifs. The sequences of the two putative PAM2motifs are also shown. The amino acid residuesthat were mutated in the experiment are shaded.(E) COS-7 cells were transfected with pMEMyc-

Tob and either pFlag-PABPC1 (lanes 2,7), pFlag--PABPC1 F567A (lanes 3,8), pFlag-PABPC1 K580A/L585A (lanes 4,9), or pFlag-PABPC1F567A/K580A/L585A (lane 5,10). (Lanes 1,6) As a control, COS-7 cells were transfected with pMEMyc and pFlag-PABPC1. The cellextracts were subjected to an immunoprecipitation assay using anti-Myc antibody. The immunoprecipitates (lanes 6–10) and theinputs (lanes 1–5, 5% of the amount immunoprecipitated) were analyzed by immunoblotting using the indicated antibodies.





HA-Pan2 showed that the CHX treatment remarkablyattenuated the interaction between PABPC1 and hPan3without affecting that between hPan3 and hPan2 (Fig.5D). Thus, CHX treatment had opposite effects on eRF3–PABPC1 and hPan3–PABPC1 interactions. These resultssuggest that a translation-dependent exchange of eRF3and hPan3 occurs on PABPC1. Tob was refractory to thisanalysis, since Tob has a quick turnover through theubiquitin–proteasome pathway and CHX treatmentgreatly affects the amount of Tob.

Deadenylating activities of both hCaf1–hCcr4and hPan2–hPan3 are stimulated by PABPC1

We demonstrated previously that the binding of PABPC1to hPan2–hPan3 leads to the activation of the deadenyl-ase (Uchida et al. 2004). In order to assess the generalityof this observation for hCaf1–hCcr4, we tested the effectof PABPC1 on the activity of hCaf1. We first preparedthe hCaf1–Tob complex. Flag-tagged hCaf1 and HA-tagged Tob were coexpressed in COS-7 cells, and the cell

extracts were subjected to immunoprecipitation withanti-Flag IgG beads. To deplete the copurified PABPC1from the hCaf1–Tob complex, the precipitated beadswere washed with buffer containing 200 mM NaCl. Thiswashing markedly attenuated the interaction betweenTob and PABPC1 without affecting the interaction be-tween hCaf1 and Tob (Fig. 6A). Thus, >85% of PABPC1could be removed from the immunopurified preparationas compared with that prepared without the high-saltwash. As shown in Figure 6B, Tob was copurified withhCaf1 as a complex (Tob–hCaf1 complex). For compari-son, we also prepared a nuclease-deficient hCaf1 mutant(D161A) as a complex with Tob (Tob–D161A complex).The RNase activities of the purified proteins were exam-ined in the presence or absence of spermidine. Figure 6Cshows that the purified Tob–hCaf1 complex could de-grade poly(A) RNA in the presence of spermidine (col-umn 5), whereas the Tob–D161A complex had no RNaseactivity (column 6). As in the case of hPan2–hPan3, noneof the proteins exhibited any detectable RNase activityin the absence of spermidine (Fig. 6C, columns 1–3). Un-der this assay condition, the effect of PABPC1 on RNase

Figure 5. Both hPan3 and Tob compete with eRF3 toform a complex with PABPC1 in a translation-depen-dent manner. (A) COS-7 cells were transfected withpFlag-hPan3 and increasing amounts of pEBG-eRF3 (1–204) (0, 3, 6, and 12 µg in lane 2, 3, 4, and 5, respec-tively). The total amounts of plasmid introduced intothe cells were maintained at the same level by cotrans-fecting the control vector. The cell extracts were sub-jected to an immunoprecipitation assay using anti-Flagantibody. (Top) Immunoblot analyses were performedwith the indicated antibodies. (Bottom) The ratios ofbound PABPC1 to precipitated Flag-hPan3 and the ex-pression level of GST-eRF3 (1–204) in the cell lysateswere measured. Error bars indicate SEM of three inde-pendent experiments. (B, top) COS-7 cells were trans-fected with pMEFlag-Tob and increasing amounts ofpcDNA3-eRF3-(His)6, and an immunoprecipitaion as-say was performed as described in A. (Bottom) The ra-tios of bound PABPC1 to precipitated Flag-Tob and theexpression level of eRF3-(His)6 in the cell lysates weremeasured. Error bars indicate SEM of three independentexperiments. (C) COS-7 cells were transfected withpFlag-CMV5 or pFlag-eRF3 and treated with or withoutcycloheximide (100 µg/mL) for 4 h. The cell extractswere subjected to immunoprecipitation assay usinganti-Flag antibody. (Top) Immunoblot analyses wereperformed with the indicated antibodies. (Bottom) Theratios of bound PABPC1 to precipitated Flag-eRF3 werecalculated. Error bars indicate SEM of at least four in-dependent experiments. (D) COS-7 cells were trans-fected with pHA-hPan2 and either pFlag-CMV5 orpFlag-hPan3, and CHX treatment and immunoprecipi-tation assay were performed as described in C.

Funakoshi et al.




activity was assessed. As shown in Figure 6D, the recom-binant GST-fused PABPC1 prepared from Escherichiacoli could markedly stimulate the RNase activity of theTob–hCaf1 complex (column 6). In sharp contrast, GST-PABPC1 could not stimulate the RNase activity of theTob–D161A complex (Fig. 6D, column 10) or hCaf1not complexed with Tob (Fig. 6D, column 4). These re-sults indicate that PABPC1 stimulates hCaf1 through itsassociation with Tob, consistent with the above findingthat Tob mediates hCaf1 binding to PABPC1. To furtherconfirm the significance of Tob in the PABPC1-stimu-lated hCaf1 activity, we also prepared hCaf1 in a com-plex with Tob whose PAM2 motifs were mutated. Asshown in Figure 6E, F139A mutation of the first PAM2motif of Tob had no significant effect on the PABPC1-stimulated RNase activity (column 4), whereas F139A/F274A mutations, which abrogated the Tob–PABPC1binding, almost abolished the effect of PABPC1. Takentogether with previous findings on hPan2–hPan3, theseresults suggest that recruitment of hPan2–hPan3 andhCaf1–hCcr4 to the poly(A)-bound PABPC1 is the gen-eral mechanism leading to the activation of the mRNAdeadenylases.

Significant roles of PABPC1-binding PAM2 motifsof Tob, hPan3, and eRF3 for mRNA deadenylationin cells

The above results strongly suggest that PAM2 motifs ofTob and hPan3 play essential roles in mRNA deadenyla-tion by recruiting hCaf1–hCcr4 and hPan2 to poly(A)-bound PABPC1. To test this possibility, we applied adominant-negative approach. T-Rex-HeLa cells weretransfected with a �-globin reporter plasmid, and thedeadenylation and decay kinetics were examined bytranscriptional pulse-chase as described in Figure 2. Asshown in Figure 7A, ectopic expression of hPan3 withPAM2 mutations (Y57A/F93A) slowed the rate of dead-enylation in the first phase compared with the controlcase. The kinetics were similar to those seen with thehPan2 D1083A mutant (Fig. 2A). In sharp contrast,ectopic expression of Tob with PAM2 mutations(F139A/F247A) reduced the rate of deadenylation in thesecond phase without affecting the rate in the first phase(Fig. 7C), with kinetics resembling those seen with thehCaf1 D161A mutant (Fig. 2B). These results provide evi-dence that Tob actually functions with hCaf1–hCcr4 in

Figure 6. Tob-mediated binding of PABPC1 leadsto the activation of hCaf1. (A) COS-7 cells weretransfected with pFlag-hCaf1 and pME-HA–Tob.The cell extracts were subjected to immunoprecipi-tation using anti-Flag IgG agarose. After washingwith buffer containing the indicated concentrationsof NaCl, proteins were eluted with the Flag peptidefrom the immunoprecipitated beads. The precipi-tated proteins were immunoblotted with the indi-cated antibodies. (B) COS-7 cells were transfectedwith pFlag-hCaf1 (lanes 2,3) or pFlag-hCaf1 D161A(lanes 4,5) and either pMEFlag (lanes 2,4) or pME-Flag-Tob (lanes 3,5). The cell extracts were subjectedto immunoprecipitation as described in A. Buffercontaining 200 mM NaCl was used for washing thebeads. (C) The purified Flag-tagged hCaf1 and hCaf1(D161A) (as shown in B), were incubated with inter-nally 32P-radiolabeled poly(A) RNA for 30 min at30°C in the presence or absence of 1 mM spermi-dine, and 32P radioactivity released was measured.(D) The purified proteins (as shown in B) were incu-bated with the 32P-radiolabeled poly(A) RNA for 30min at 30°C with or without 10 ng of GST-fusedPABPC1 in the absence of spermidine. (E, left)COS-7 cells were transfected with pFlag-hCaf1 andeither pMEHA–Tob (lane 1), pMEHA–Tob (F139A)(lane 2), or pMEHA–Tob (F139A/F274A) (lane 3).The cell extracts were subjected to immunoprecipi-tation as described in B. (Right) The purified pro-teins were incubated with the 32P-radiolabeledpoly(A) RNA as described in D.





the second phase of mRNA deadenylation, and furthersupport the notion that PAM2 motifs play essential rolesin triggering mRNA deadenylation. We also examinedthe effect of PAM2 mutations in eRF3 (F66A/F75A),which abrogated the binding to PABPC1 (SupplementaryFig. 3). Ectopic expression of the mutant slowed the rateof deadenylation mainly in the second phase (Fig. 7E).These results indicate that PAM2 motifs of Tob, hPan3,and eRF3—namely, PABPC1 binding—play key roles ineRF3-mediated mRNA deadenylation.

Discussion

We demonstrated previously that eRF3 mediates dead-enylation and decay of normal mRNA in a mannercoupled to translation termination. In this study, weclarified the underlying mechanism of the eRF3-medi-ated mRNA deadenylation. The main findings of thisstudy are (1) eRF3 mediates deadenylation throughPan2–Pan3 and Caf1–Ccr4 deadenylases, which are con-served from yeast to humans (Figs. 1, 2); (2) the termina-tion complex eRF1–eRF3 and deadenylase complexeshPan2–hPan3 and hCaf1–hCcr4 interact with PABPC1,in all cases via PAM2–PABC contacts (Figs. 3, 4); (3)hPan2–hPan3 binds PABPC1 competitively with eRF3and in a manner dependent on translation (Fig. 5); and (4)the binding of hPan2–hPan3 and hCaf1–hCcr4 with

PABPC1 leads to the activation of the deadenylase activ-ity (Fig. 6; Uchida et al. 2004). Together, these new find-ings define PABPC1 as a regulatory platform for mRNAdeadenylation, and argue that the recruitment of dead-enylating enzymes to PABPC1 is involved in the mecha-nism of activation of mRNA deadenylation. The datasuggest that eRF3 mediates mRNA deadenylation bytriggering the recruitment of the deadenylase complexesto PABPC1; as translation proceeds, the terminationcomplex is released from PABPC1 and in turn the dead-enylase complexes bind to PABPC1, which leads to theactivation of the deadenylating enzymes.

General mechanism of mRNA deadenylation

Several previous reports using specific mRNAs showedthat recruitment of deadenylating enzymes to themRNA is involved in the mechanism of mRNA dead-enylation. First, in Drosophila embryos, an RNA-bind-ing protein, Smaug, which physically interacts with theCaf1–Ccr4 complex, recruits the complex to Hsp83mRNA to trigger deadenylation and decay of the tran-script. The recruitment-induced enzyme activation wasconfirmed by tethering Smaug to reporter transcripts (Se-motok et al. 2005). Second, a similar tethering strategywas used to show that selective recruitment of Caf1–Ccr4 is sufficient to activate mRNA decay in yeast (Fi-

Figure 7. hPan3, Tob, and eRF3 with mutationsin the PABPC1-binding PAM2 motifs affectmRNA deadenylation in a dominant-negativefashion. (A) T-Rex-HeLa cells were transfectedwith pFlag-CMV5/TO-G1 reporter and eitherpHA-CMV5 (lanes 1–5) or pHA-hPan3 Y57A/F93A (lanes 6–10). Transcriptional pulse-chaseanalysis was performed as described in Figure 2.(B) Total cell lysates were analyzed by immuno-blotting using anti-HA (top) and anti-GAPDH(bottom). (C) As in A, except that the cells weretransfected with pMEHA or pMEHA–TobF139A/F274A, and transcriptional pulse-chaseanalysis was performed. (D) As in B, total celllysates were immunoblotted. (E) As in A, exceptthat the cells were transfected with pFlag-CMV5or pFlag-eRF3 F66A/F75A, and transcriptionalpulse-chase analysis was performed. (F) As in B,except that anti-Flag antibody was used, and to-tal cell lysates were immunoblotted.

Funakoshi et al.




noux and Seraphin 2006). Third, yeast Mpt5, a memberof the PUF family of RNA-binding proteins, also recruitsthe Caf1–Ccr4 complex to HO mRNA to trigger dead-enylation of the message (Goldstrohm et al. 2006).Fourth, an RNA-binding protein, CUG-BP, specificallybinds to ARE-containing mRNAs to stimulate poly(A)shortening by recruiting another poly(A)-specific ribo-nuclease, PARN, to the mRNA (Moraes et al. 2006). Forthe hPan2–hPan3 deadenylase complex, we demon-strated previously that hPan3 recruits the hPan2 cata-lytic subunit to poly(A)-bound PABPC1 to activate itspoly(A) nuclease activity (Uchida et al. 2004), which is inaccord with a previous report showing that Pan2–Pan3 isa Pab1-dependent poly(A) nuclease in yeast (Brown et al.1996). In this study, we further demonstrated thathCaf1–hCcr4 is also activated by its recruitment topoly(A)-bound PABPC1.

Our results indicate that anti-proliferative protein Tobis a key component of hCaf1–hCcr4. We presented threeimportant observations showing physical and functionalinteraction between hCaf1–hCcr4 and PABPC1, which issupported by structural evidence. First, hCaf1 indirectlyinteracts with PABPC1 through Tob (Fig. 4A,B). Second,the binding of PABPC1 leads to the stimulation of hCaf1deadenylase (Fig. 6). Third, the binding requires thePAM2 motif of Tob and the PABC domain of PABPC1(Fig. 4D,E). The PAM2–PABC contact is also utilized forthe interaction between hPan3 and PABPC1 (Fig. 3D,E).Thus, recruitment of both of the two major mRNA dead-enylase complexes to poly(A)-bound PABPC1 throughadaptor proteins is an important determinant in the ac-tivation of the deadenylases. These results suggest thebiological significance of the mechanism of activation ofmRNA deadenylases by PABPC1 in the regulation ofmRNA deadenylation. One possible role of the adaptorproteins (Tob and hPan3) is to recruit mRNA deadenyl-ase specifically to the substrate mRNA poly(A) tails bytaking advantage of the poly(A) specificity of PABPC1. Inthe case of hPAN, the adaptor protein hPan3-mediatedPABPC1 interaction actually increases its substratespecificity for poly(A) RNA (Uchida et al. 2004). The sec-ond possible role is to reduce entropy in the reaction byplacing the deadenylase in close proximity to the sub-strate RNA. In the case of both deadenylases, >10-foldstimulation of the enzyme activity was observed whencomplexed with PABPC1 through the adapter.

The role of translation termination factor eRF3in mRNA deadenylation

Our previous and present results show that eRF3 medi-ates deadenylation and decay of normal mRNA in a man-ner coupled to translation termination and the eRF3-me-diated deadenylation is catalyzed by both hCaf1–hCcr4and hPan2–hPan3. As translation proceeds, eRF3 is re-leased from PABPC1, and hPan2–hPan3 (and possiblyhCaf1–hCcr4) associates with PABPC1, which leads tothe activation of the deadenylases. Thus, eRF3 triggersdeadenylation by recruiting the deadenylases to poly(A)-bound PABPC1. Since eRF3 competes with the deadenyl-

ases for PABPC1 binding, dissociation of eRF3 fromPABPC1 is likely to be the key determinant in triggeringdeadenylation. As in the case for other G protein familymembers, eRF3 is thought to function as a molecularswitch that changes binding partners, depending onwhether it is in its GTP- or GDP-bound form. Since ri-bosome and eRF1 stimulate GTP hydrolysis of eRF3(Frolova et al. 1996), a conformational change of eRF3during or after translation termination might trigger dis-sociation of eRF3 from PABPC1. Consistent with thisidea, recent findings suggested that binding of PABPC1and eRF1 to eRF3 might be mutually exclusive (Hau-ryliuk et al. 2006).

Our present study has focused on the role of eRF3 innormal mRNA decay. However, eRF3 also functions inNMD. NMD is a surveillance mechanism that degradesmRNA containing premature termination codons(PTCs). Since PTCs are termination codons, recognitionof PTCs is thought to be mediated by translation termi-nation. Recent findings demonstrated that eRF1–eRF3binds the NMD factors SMG1–Upf1 to form a complextermed SURF, and an association between SURF and theexon junction complex triggers phosphorylation of Upf1(Kashima et al. 2006). Although it is not known howUpf1 phosphorylation leads to rapid decay of PTC-con-taining mRNA, eRF3 must play a pivotal role in trigger-ing NMD.

We and others showed previously that a member of theeRF3 family of G proteins, Ski7, interacts with the exo-some to function in 3�-to-5� decay of mRNA (van Hoof etal. 2000; Araki et al. 2001), and later, Ski7 was shown tobe involved in the decay of aberrant mRNAs lacking ter-mination codons, which is called “nonstop mRNA de-cay” (NSD) (Frischmeyer et al. 2002; van Hoof et al.2002). In NSD, Ski7 is suggested to bind to a ribosomestalled at the 3� end of the mRNA and recruit the exo-some to the mRNA to trigger 3�-to-5� decay of themRNA. In addition, Hbs1, the closest relative of eRF3,has similar roles in another type of aberrant mRNA.mRNAs with ribosomes stalled before translation termi-nation are recognized and degraded by a pathway referredto as “no-go decay” (NGD) (Doma and Parker 2006). InNGD, Hbs1 is suggested to recognize the stalled ribo-some to trigger endonucleolytic cleavage of the mRNA.Thus, eRF3 family members play key roles in triggeringmRNA decay by functioning as a molecular switch con-necting translation to mRNA decay.

Future challenges include clarifying the mechanism ofsequential deadenylation by hPan2–hPan3 and hCaf1–hCcr4 during poly(A) tail shortening of mRNA. As re-ported previously, our results also show that the poly(A)tail is first shortened in a distributive manner by hPan2–hPan3 to ∼100 nt (first phase, 0–3 h), and the remainingpoly(A) tail is then rapidly and processively degraded byhCaf1–hCcr4 (Figs. 2, 7; Yamashita et al. 2005). It is notclear, however, why eRF3 triggers activation of hPan2–hPan3 in the first phase, but hCaf1–hCcr4 in the secondphase. Recent work by Keeling et al. (2006) may providea clue to this question. They found a novel factor (termedTpa1) that influences translation termination and





mRNA deadenylation in S. cerevisiae. Tpa1 interactswith both eRF1–eRF3 and Pab1, and is suggested to beinvolved in the termination-coupled mRNA deadenyla-tion (Keeling et al. 2006). Interestingly, Tpa1 serves as apositive regulator of Pan2–Pan3 and a negative regulatorof Caf1–Ccr4. Thus, Tpa1, through the interaction witheRF3, might positively and negatively regulate the re-cruitment of Pan2–Pan3 and Caf1–Ccr4 to Pab1, respec-tively (Keeling et al. 2006). Further studies will be re-quired to test this possibility.

Note

During preparation of our manuscript, there appeared apaper (Siddiqui et al. 2007) in which the authors report aphysicochemical analysis of PAM2–PABC contact be-tween hPan3 and PABPC1 by using NMR. They showedthat the interaction is conserved from yeast to humans,and is involved in the first trimming of mRNA poly(A)tails in yeast.

Materials and methods

Plasmids

To construct p9L-MFA2pG with the MFA2pG gene under thecontrol of the tetO promoter, the cassette harboring the tetOpromoter and CYC1 terminator was excised from pCM190 andinserted into YEplac181 to give p9L. MFA2pG fragment wasexcised from pGAL-MFA2pG (Araki et al. 2001) and subclonedinto p9L. To construct pEBG-eRF3 (1–204) and pCMV-Myc-eRF3 N (1–204), the corresponding cDNA fragments of eRF3were inserted in pEBG and pCMV-Myc (Clontech), respectively.In this study, we used the GSPT2 gene for the plasmids express-ing eRF3. To construct pFlag-PABP�C, a PCR fragment encod-ing the N-terminal 375 amino acids of PABPC1 was inserted inpFlag-CMV5 (Eastman Kodak Co.). To construct pMEFlag-Toband pFlag-Caf1, the full-length Tob and hCAF1 cDNAs wereamplified by RT–PCR using total RNA from HeLa cells andinserted into pMEFlag (a Flag-tagged vector based onpME18SFL3) and pFlag-CMV5, respectively. pMEHA-Tob andpHA-CAF1, which contain the HA tag instead of Flag, were alsoconstructed. To generate pMEFlag-Tob (1–218) and pMEFlag-Tob (110–345), the corresponding cDNA fragments of Tob wereamplified by PCR and inserted into pMEFlag. Point mutants inhCaf1, PABPC1, hPan3, Tob, and eRF3 were generated frompHA-hCaf1, pFlag-PABPC1, pHA-hPan3, pMEFlag-Tob andpFlag-eRF3, respectively, using a QuickChange Site-DirectedMutagenesis Kit and were confirmed by sequencing. pFlag-CMV5/TO-G1 was generated from pFlag-CMV5 by insertingthe two Tet-operators and the �-globin gene downstream fromits CMV promoter. Construction of all other plasmids used inthis study has been described previously (Hosoda et al. 2003;Uchida et al. 2004).

Yeast strains and growth conditions

The yeast strains used were derived from MBS (MATa ade2 his3leu2 trp1 ura3 can1) and are listed in Supplementary Table 1.Strains were grown in standard media. Disruption of yeast geneswas achieved using standard PCR-based methods and the dis-ruption was confirmed by PCR reactions.

Cell culture and DNA transfection

COS-7, HEK-293T, and T-REx-HeLa cells (Invitrogen) were cul-tured in Dulbecco’s modified Eagle’s medium (Nissui) supple-mented with 10% fetal calf serum. Cells were transfected withplasmid using Lipofectin or LipofectAMINE 2000 (Invitrogen).

Immunoprecipitation

The transfected cells were lysed with 10 µg/mL of boiledRNaseA in buffer A consisting of 20 mM Tris-HCl (pH 8.0),50 mM NaCl, 1% Nonidet P-40, 1 mM dithiothreitol (DTT),2.5 mM EDTA-Na (pH 8.0), 100 µM PMSF, 2 µg/mL of aproti-nin, and 2 µg/mL of leupeptin. After centrifugation at 15,000gfor 20 min, the lysate was incubated for 1 h at 4°C with anti-FlagIgG agarose (Sigma) or Anti-HA Affinity Matrix (Roche Diag-nostics), and then the resin was washed with buffer A. Asneeded, recombinant proteins were added and the resin wasfurther incubated for 60 min at 4°C. After washing with bufferA, proteins retained on the resin were subjected to SDS-PAGEand immunoblot analysis. The antibodies used for immunob-lotting were anti-Flag M2 (Sigma), anti-HA (3F10, Roche Diag-nostics), anti-Myc (9E10 [Sigma] or A-14 [Santa Cruz Biotech-nology]), anti-GST (Santa Cruz Biotechnology), anti-His (MBL),anti-Tob (clone 4B1, Sigma), anti-PABP (kind gift from H.Imataka and N. Sonenberg), and anti-GSPT (Hoshino et al.1998).

RNA analysis

For the Tet-off transcriptional repression experiment, yeastcells were grown in selective medium, which contained 2%galactose to produce the Sup35 N domain, for 6 h at 30°C (yeastcells reached mid-log phase [OD600 = 0.5–0.6] in this condition).Cells were collected by centrifugation and resuspended in 10mL of selective media. After the cells were incubated for 10 minat 30°C, doxycycline was added to a final concentration of 5µg/mL to repress transcription. At the indicated times, cellswere harvested and immediately frozen in liquid nitrogen. Forpreparation of total RNA in the steady state, yeast cells weregrown to mid-log phase (0.5–0.6) and collected by centrifuga-tion. RNA extraction from the cell pellet was performed as de-scribed previously (Hosoda et al. 2003). To determine thepoly(A) tail length of MFA2pG mRNA, total RNA was separatedon a 4% polyacrylamide/8 M urea gel and electroblotted to Hy-bond XL (Amersham Pharmacia). For the analysis of mRNAdecay, total RNA was separated on a 1% agarose gel and blottedto Hybond XL as described previously. The reporters were de-tected by Northern blotting using an end-labeled oligonucleo-tide, oRP121 (5�-AATTCCCCCCCCCCCCCCCCCCA-3�),which is complementary to the poly(G) sequence at the 3�-un-translated region of the transcript, as a probe. The poly(A) taillength analysis for total RNA was performed as described pre-viously (Brown et al. 1996).

For the transcriptional pulse-chase analysis, T-Rex-HeLa cellswere transfected with pFlag-CMV5/TO-G1. After 24 h, cellswere treated with 10 ng/mL tetracycline for 2.5 h to inducetranscription, and were harvested at the specified time aftertranscription shutoff. Total RNA was isolated with TriPure Re-agents (Roche) and separated by 2.2% agarose gel electrophore-sis using a NorthernMax Kit (Ambion). �-Globin mRNA wasdetected by Northern blotting using [32P]-labeled �-globin ORFas a probe.

In vitro nuclease assay

The production of internally 32P-radiolabeled poly(A) RNA wasdescribed previously (Uchida et al. 2004). In brief, oligo(A)23 was

Funakoshi et al.




extended using [�-32P]ATP and yeast poly(A) polymerase (USBCorp.). The labeled poly(A) RNA was purified by gel filtrationwith S-400 HR MicroSpin columns (Amersham Bioscience) andethanol precipitation. For the immunopurification of recombi-nant proteins, extracts prepared from COS-7 cells transfectedwith pFlag-hCAF1 and with either pMEHA or pMEHA-Tobwere immunoprecipitated with anti-Flag antibody in buffer A.After washing three times with buffer A, the precipitated pro-teins were eluted with buffer B (10 mM HEPES-Na at pH 7.5, 2mM MgCl2, 1 mM DTT, 0.02% Nonidet P-40) containing 100µg/mL Flag peptides (Sigma). The immunopurified proteinswere incubated for 30 min at 30°C in buffer B with 1 U/µLRNasin (Promega) and 1 × 105 counts per minute (cpm) of la-beled poly(A) RNA in the presence or absence of 1 mM spermi-dine. If necessary, recombinant GST-fused PABPC1 was addedto the reactions. The extent of nucleolysis was measured asfollows: Aliquots were precipitated with 25% trichloroaceticacid by centrifugation at 20,000g, and the radioactivity of thesoluble fraction was measured with a liquid scintillationcounter. Purification of recombinant GST-PABPC1 was per-formed as described previously (Uchida et al. 2004)

Acknowledgments

We thank Dr. Kazuyoshi Yonezawa and Dr. Tadashi Yamamotofor plasmids. This work was supported in part by research grantsfrom the Japanese Ministry of Education, Culture, Sports, Sci-ence and Technology, and Grant-in-Aid for Research in NagoyaCity University.

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