Zygotic Control of Maternal Cyclin A1 Translation andmRNA StabilityYANN AUDIC,1 MARK GARBRECHT,1 BRIAN FRITZ,2 MICHAEL D. SHEETS,2 AND

REBECCA S. HARTLEY1*1Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa2Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin

ABSTRACT Cyclin mRNAs are unstable inthe adult cell cycle yet are stable during the first12 cell divisions in Xenopus laevis. We recentlyreported that cyclin A1 and B2 maternal mRNAsare deadenylated upon completion of the 12thdivision (Audic et al. [2001] Mol. Cell Biol. 21:1662–1671). Deadenylation is mediated by the 3�untranslated region (UTR) of the mRNA and pre-cedes the terminal disappearance of the cyclinproteins, with both processes requiring zygotictranscription. The purpose of the current studywas (1) to ask whether deadenylation leads totranslational repression and/or destabilizationof endogenous cyclin A1 and B2 mRNAs, and (2)to further characterize the regulatory sequencesrequired. We show that zygote-driven deadenyla-tion leads to translational repression and mRNAdestabilization. A 99-nucleotide region of the3�UTR of the cyclin A1 mRNA mediates bothdeadenylation and destabilization. Surprisingly,two AU-rich consensus elements within this re-gion are dispensable for this activity. These re-sults suggest that zygote-dependent deadenyla-tion, translational repression, and mRNAdestabilization by means of novel 3�UTR ele-ments contribute to the disappearance of mater-nal cyclins. They also suggest that translationalcontrol of cyclins may play a role in the transi-tion to the adult cell cycle. These data concurwith previous studies in Drosophila showing thatzygote-mediated degradation of maternal cdc25mRNA may be a general mechanism wherebytransition to the adult cell cycle proceeds.© 2002 Wiley-Liss, Inc.

Key words: Cyclin A1 mRNA; translational con-trol; deadenylation; 3�UTR; cell cy-cle; Xenopus Laevis

INTRODUCTION

Translation of mRNAs at the correct time and placeis essential for early embryogenesis of both vertebratesand invertebrates. Maternally synthesized mRNAsand proteins are stored in the developing oocyte anddrive meiosis, early embryonic cell divisions, cell fatedecisions, and embryonic patterning (Ambros, 2000;Lasko, 2000; Bashirullah et al., 2001; de Moor and

Richter, 2001). The poly(A) tail length often influencestranslation of maternal mRNAs (Wickens et al., 1997;de Moor and Richter, 2001). The binding of proteins toregulatory sequences in the 3� untranslated region(3�UTR) of the mRNA is one way that poly(A) taillength is altered.

In maturing Xenopus laevis (frog) and mouse oocytes,mRNAs necessary for meiosis are specifically polyade-nylated and translationally activated. Among thesemRNAs are those encoding tPA, c-mos, cyclin B1, andcyclin A1 (Sheets et al., 1994, 1995; Stutz et al., 1998).Elongation of the poly(A) tail depends on the nuclearpolyadenylation sequence AAUAAA (NPS) and UA-rich sequences termed either adenylation control ele-ments (ACEs) for mice or cytoplasmic polyadenylationelements (CPEs) for frogs (Huarte et al., 1992; Hakeand Richter, 1994; Stebbins-Boaz et al., 1996; Stutz etal., 1998; Mendez and Richter, 2001). In frog oocytes,polyadenylation is initiated when the CPE binding pro-tein (CPEB) is phosphorylated and recruits the cleav-age and polyadenylation specificity factor (CPSF) to theNPS (Mendez et al., 2000). Translational activationoccurs when Maskin, a protein binding to both CPEBand the cap binding protein eIF4E, dissociates fromeIF4E, allowing the binding of eIF4G and positioningof the 40S ribosomal subunit (Mendez and Richter,2001).

After fertilization, the frog embryo is transcription-ally inactive and translational control of maternalmRNAs continues to be a dominant regulator of geneexpression (Paris and Philippe, 1990; Richter, 1999).The poly(A) tails of cyclin mRNAs are further elon-gated after fertilization, presumably due to the CPEsin their 3�UTRs (Sheets et al., 1994). C-rich elementshave been shown recently to control poly(A) addition ofthe maternal mRNA encoding the phosphatase PP2A

Grant sponsor: American Cancer Society; Grant number: RSG-01-054-01-CCG.

Dr. Audic’s current address is UMR 6061-CNRS, Genetique et De-veloppement, Universite de Rennes I, France.

*Corresponding author: Rebecca S. Hartley, Department of CellBiology and Physiology, University of New Mexico, Health SciencesCenter, Albuquerque, NM 87131. E-mail: rhartley@salud.unm.edu

Received 27 June 2002; Accepted 4 September 2002DOI 10.1002/dvdy.10191

DEVELOPMENTAL DYNAMICS 225:511–521 (2002)

© 2002 WILEY-LISS, INC.

(Paillard et al., 2000), whereas BMP-7 mRNA is poly-adenylated and translated in early embryos eventhough its 3�UTR does not contain known polyadenyl-ation elements (Fritz and Sheets, 2001). The embryonicdeadenylation element (EDEN), an UG/UA rich se-quence, and its binding protein EDEN-BP trigger thedeadenylation of several maternal mRNAs immedi-ately after fertilization, including c-mos and c-jun(Paillard et al., 1998, 2002). AU-rich elements (AREs),which destabilize cytokine mRNAs in somatic cells,also act as deadenylation elements in early frog em-bryos (Voeltz and Steitz, 1998). In Drosophila, the RNAbinding proteins Pum and Nos establish embryonicpolarity by promoting posterior deadenylation ofHunchback mRNA (Wreden et al., 1997). Pum and Nosmay also translationally repress cyclin B mRNA in thepole cells of Drosophila embryos (Sonoda and Wharton,2001). All of the above elements control development ofthe transcriptionally silent embryo.

The mechanism by which deadenylation results intranslational repression is not well-characterized. Ithas been shown that the frog homolog of Pum interactswith CPEB, suggesting that Pum may repress transla-tion of cyclin B by means of this interaction (Nakahataet al., 2001) but the link to adenylation state is notknown. Poly(A) tails are bound by poly(A)-binding pro-teins (either PABP1 or ePAB) that in turn interact withthe translation initiation factor eIF4G, presumably ac-tivating translation (Imataka et al., 1998). Deadenyla-tion would lead to the disruption of the PABP/eIF4Ginteraction and to a decrease in translational efficiency(Wakiyama et al., 2000). In growing mouse oocytes,tPA mRNA is partially deadenylated (from 250 down to40 A’s) upon binding of ACEB to the 45-nucleotide (nt)ACE. Deadenylation translationally silences thismRNA in the resting oocyte, and ACEB is required tomaintain dormancy (Stutz et al., 1998). Resumption ofmeiosis results in either modification or displacementof ACEB and subsequent translational activation andpolyadenylation. In this case, the short poly(A) tailappears to be mandatory for translation (Stutz et al.,1998). In addition to tPA, both c-mos (Gebauer et al.,1994) and hypoxanthine phosphoribosyl transferase(Paynton and Bachvarova, 1994) are regulated by ad-enylation status in mouse oocytes. It has been sug-gested that deadenylation in mouse oocytes may not beprerequisite for translational silencing, because a poly-adenylated ACE-containing mRNA is transientlytranslated in primary oocytes, being silenced whendeadenylation is still ongoing (Huarte et al., 1992).

Deadenylation of maternal mRNAs has been linkedto destabilization in the early embryo. In Xenopus, thebulk of zygotic transcription is activated after comple-tion of the 12th cell cycle (Newport and Kirschner,1982). With the activation of transcription, many pre-viously deadenylated maternal mRNAs are destabi-lized (Duval et al., 1990).

We recently reported that cyclin A1 and B2 maternalmRNAs are deadenylated in Xenopus embryos in a

manner dependent on zygotic transcription (Audic etal., 2001). Deadenylation precedes the terminal disap-pearance of maternal cyclin A1 and B2 proteins and theremodeling of the rapid embryonic cell cycle to thelonger adult cell cycle. The 3�UTR of either of thesecyclin mRNAs confers deadenylation on a chimericmRNA in a manner dependent on zygotic transcription.The purpose of the current study was (1) to askwhether zygote-mediated deadenylation leads to trans-lational repression and/or destabilization of endoge-nous cyclin A1 and B2 mRNAs, and (2) to furthercharacterize the regulatory sequences required. Ourresults suggest that zygote-dependent deadenylationcontributes to the disappearance of maternal cyclins byleading to translational repression and mRNA desta-bilization and, thus, may play a role in establishmentand regulation of the adult somatic cell cycle in Xeno-pus.

RESULTSDeadenylation of Cyclin A1 Maternal mRNARequires Zygotic Transcription

We showed previously that the maternal mRNA en-coding cyclin A1 is deadenylated beginning approxi-mately 6 hr postfertilization (hpf; Audic et al., 2001).This timing corresponds to the midblastula transition(MBT) when zygotic transcription begins. When a chi-meric mRNA containing the cyclin A1 3�UTR is in-jected into two-cell embryos, this mRNA is deadeny-lated with kinetics identical to that of the maternalcyclin A1 mRNA, suggesting that all the necessaryinformation is contained within the cyclin A1 3�UTR.Blocking the onset of zygotic transcription in the em-bryo abolishes deadenylation of the chimeric mRNA,suggesting that a zygote-derived product (either RNAor protein) is required for deadenylation to occur.

Our first aim was to determine whether deadenyla-tion of the endogenous maternal mRNA depends onzygotic transcription. This determination would pro-vide evidence for a common mechanism in deadenyla-tion of both chimeric and endogenous mRNA. Weblocked the initiation of transcription by injecting�-amanitin, an inhibitor of RNA polymerase II, intoone-cell embryos and assayed the poly(A) tail length ofcyclin A1 mRNA by poly(A) tail (PAT) analysis. Em-bryos were injected with �-amanitin or its buffer (con-trol) at the one-cell stage (1 hpf), and RNA was ex-tracted from developing embryos for both PAT andNorthern analyses. Reverse transcriptase-polymerasechain reaction (RT-PCR) -based PAT analysis usesoligo-d(T) and a mRNA-specific primer within the3�UTR to obtain a PCR product whose length changesdepending on the size of poly(A) tail. The position of thecyclin A1-specific primer is underlined in the 3�UTRsequence shown in Figure 1A. Results from a represen-tative PAT experiment are shown in Figure 2A. Figure2B shows Northern analysis for the zygotic transcriptGS17 (Krieg and Melton, 1985) to control for inhibitionof zygotic transcription. GS17 appears by 8 hpf in con-

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trol embryos and is not detected in �-amanitin–injectedembryos.

The expected size of a cyclin A1 product with a min-imal detectable poly(A) tail of 12 residues is 510 nt.Between 2 to 6 hpf, the cyclin A1 product in bothcontrol and �-amanitin–injected embryos migrates be-tween 600 and 700 nt, indicating a poly(A) tail of be-tween 100 and 200 residues. During this time, cyclinA1 protein is degraded and reaccumulates every 25–30min (Hartley et al., 1996), consistent with the mRNAbeing polyadenylated and efficiently translated. In con-trol embryos, cyclin A1 mRNA is rapidly deadenylatedafter 6 hpf as shown by a rapid decrease in the size of

the PCR product. Product at the minimal expected sizeis present by 8 hpf, with all of the PCR product migrat-ing at the minimal size by 10 hpf. This finding indicatesthat only a short stretch of A’s, sufficient for RT-prim-ing, is present on the mRNA. In contrast, the PCRproduct in �-amanitin–injected embryos decreases insize much more slowly and remains distributed abovethe minimal expected size even at 10 hpf. This slowerand less complete deadenylation is most likely due todefault deadenylation, which is not blocked by inhibit-ing transcription. In addition, there is a correlationbetween adenylation state of the mRNA and presenceof cyclin A1 protein. Cyclin A1 protein disappears incontrol embryos after 8.5 hpf but is present until at

Fig. 1. A: Nucleotide sequence of the Xenopus cyclin A1 3� untrans-lated region cloned into the pGbORF vector. The 3�untranslated region(3�UTR) of cyclin A1 (accession no. X53745) encompasses nucleotides(nt) 1318 to 1813. The last 12 nt of the 3�UTR were not cloned, and theXbaI and EcoRV sites were added by polymerase chain reaction. Thenuclear polyadenylation sequence AAUAAA (NPS) is italicized, cytoplas-mic polyadenylation elements (CPEs) are in bold italics, and AU-richelements (AREs) in capital letters. Restriction sites are indicated. The 5�forward poly(A) tail (PAT) primer anneals to nt 1318-1335 (indicated byunderlining). The ARE sequence deleted in the GbA1�SE-�ARE is de-limited by arrowheads. B: Schematic of chimeric mRNAs. The �-globin5�UTR, open reading frame, and partial 3�UTR are indicated (GbORF)and were present in all chimeric mRNAs. GbA1 contains the completecyclin A1 3�UTR as shown in A. Restriction sites are indicated as (X)XbaI, (S) SpeI, (H) HpaI, and (E) EcoRV. The black rectangle representsthe general location of the CPE and hexanucleotide. Dashed lines indi-cate the region deleted from the 3�UTR. GbA1�XS has the region be-tween XbaI and SpeI deleted, and GbA1�XH has the region betweenXbaI and HpaI deleted. GbA1�HE has the region between HpaI andEcoRV deleted, and GbA1�SE has the region between SpeI and EcoRVdeleted. GbA1�SE-�ARE has nt 1417-1499 deleted in addition to the�SE. GbA1�384 contains only the proximal 99 nt (1317-1416) of the3�UTR. GbA1�HE, GbA1�SE, GbA1�SE-�ARE, and GbA1�384 havean exogenous (histone B4) CPE and NPS added (indicated by rectanglelabeled CPE) to ensure that in vivo adenylation will occur upon injection.

Fig. 2. Rapid deadenylation of cyclin A1 mRNA requires zygotictranscription. One-cell Xenopus embryos were injected with 50 ng of�-amanitin or its buffer (Control). Total RNA was extracted at the indi-cated times after fertilization and subjected to either reverse transcrip-tase-polymerase chain reaction–based poly(A) tail (PAT) analysis byusing a cyclin A1–specific primer and oligo-d(T) or to Northern blotanalysis. A: Representative DNA gel of PAT cDNA products. The mark-ers are a 100-bp DNA ladder; -RT and -cDNA are controls omitting eitherthe reverse transcriptase or the cDNA template. The arrowhead indicatesthe expected size for a cyclin A1–specific product with a minimal poly(A)tail (510 nucleotides). B: Northern blot for the zygote-specific mRNAGS17. C: Imagequant was used to quantify the signal in each lane, andthe results of three separate experiments were averaged. The position(distance from the A12 signal � minimum predicted size) of the peaksignal was determined and plotted to determine the poly(A) tail size of theendogenous RNA. The distance of the slowest migrating band was setequal to 100. hpf, hours postfertilization.

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least 11 hr in �-amanitin–injected embryos, indicatingthat this mRNA is still translatable (Audic et al., 2001).

The position (distance from the A12 signal � mini-mum predicted size) of the peak signal was determinedand plotted using Imagequant, to determine thepoly(A) tail size of the endogenous RNA. Figure 2Csummarizes these data and clearly shows the differ-ence in deadenylation kinetics in control vs. �-aman-itin embryos. These results show that rapid deadeny-lation of endogenous maternal cyclin A1 mRNA afterthe MBT requires synthesis of a zygotic product, asdoes the deadenylation of chimeric RNA containing thecyclin A1 3�UTR. Thus, the same mechanism likelycontrols the adenylation/deadenylation of injected andendogenous mRNA.

Release of Cyclin A1 mRNA From thePolysomes Accompanies Deadenylation

To test whether translation of cyclin A1 mRNA isaffected by deadenylation, we quantitatively analyzedits association with polysomes. Cytoplasmic extractswere prepared from pre-MBT stage 7 (4 hpf) and post-MBT stage 10 (9 hpf) embryos, when cyclin A1 mRNAshould have the maximum and minimum poly(A) taillength, respectively. Extracts were separated into poly-somal (P) and nonpolysomal fractions (S) on sucrosegradients by using standard methods (Fritz andSheets, 2001). Each fraction was then analyzed byquantitative Northern blots to detect cyclin A1 or forcomparison, cyclin B2 or B1 mRNA (Fig. 3). All three ofthe cyclin mRNAs are associated with the polysomes(P) in stage 7 embryos when transcription is still silent.The presence of cyclin mRNAs in the polysomal frac-tion is consistent with the polyadenylation of thesemRNAs at this time (compare with 4 hpf embryos in

Fig. 2; Sheets et al., 1994) and with the synthesis ofcyclin protein (Sheets et al., 1994; Audic et al., 2001).In contrast, in stage 10 embryos (post-MBT), cyclin A1mRNA is in the nonpolysomal supernatant fraction (S),as is the majority of cyclin B2 mRNA. Both of thesematernal mRNAs are deadenylated by this time (Audicet al., 2001). In contrast, cyclin B1 mRNA retains apoly(A) tail and is still present primarily in the polyso-mal fraction. The authentic association of thesemRNAs with the polysomes was verified by disruptingthe interaction with ethylenediaminetetraacetic acid(�EDTA).

These data show that after the MBT, cyclin A1mRNA is no longer translated. Polysome dissociationalso occurs for cyclin B2, another maternal cyclinwhose mRNA is deadenylated and protein terminallydown-regulated after the MBT. In contrast, cyclin B1mRNA is not specifically deadenylated, does not disso-ciate completely from the polysomes, and the proteinpersists in post-MBT embryos (Hartley et al., 1996;Audic et al., 2001). These are the first data showingthat the terminal disappearance of cyclin A1 in devel-oping embryos is due to translational repression aswell as protein instability.

mRNA Destabilization Follows Deadenylationand Release From the Polysomes

To determine whether deadenylation and releasefrom the polysomes is followed by cyclin A1 mRNAdestabilization, we performed Northern analysis onRNA isolated from control and transcriptionally inhib-ited embryos. Transcriptional inhibition was verifiedby reprobing the blot for GS17. Equivalent RNA load-ing was assayed by probing for ornithine decarboxylase(ODC) mRNA. ODC mRNA is present at constant lev-els and is stably polyadenylated and translated duringthis time (Osborne et al., 1991). Relative mRNA levelswere quantitated by using Imagequant software.

Figure 4 shows that the cyclin A1 mRNA level re-mains fairly constant through 8 hpf in control embryos.By 10 hpf, the amount of cyclin A1 mRNA has de-creased significantly, with less than 20% of the mater-nal mRNA remaining at 10 hpf and less than 5% by 14hpf. Destabilization of the mRNA at 10 hpf followedrelease of the mRNA from polysomes at 9 hpf (Fig. 3)and only occurred after the initiation of zygotic tran-scription, as shown by GS17 expression. In the absenceof zygotic transcription (�-amanitin), cyclin A1 mRNAlevel remains constant until 8 hpf, with only a slightdecrease occurring after this time. By 14 hpf, approxi-mately 50% of the original maternal mRNA is stillpresent under �-amanitin conditions. This slow de-crease does not appear to be specific for cyclin A1 asODC mRNA also decreases during this time. Thus,deadenylation and release from the polysomes is fol-lowed by destabilization of cyclin A1 mRNA, ensuringits translational silencing.

Fig. 3. Cyclin A1 mRNA dissociates from the polysomes after themidblastula transition. Polysomal (P) and nonpolysomal (S) fractionswere prepared from stage (st.) 7 (4 hr postfertilization [hpf]) and stage 10(9 hpf) Xenopus embryos. Total RNA was isolated from unfractionatedsamples (T), the polysomal pellet (P), and the nonpolysomal supernatant(S), and analyzed by Northern blot hybridization. cDNA probes were usedto detect cyclin A1, cyclin B2, and cyclin B1 mRNAs. A representativeNorthern blot for two stages of development is shown, along with theethylenediaminetetraacetic acid (EDTA) control experiment. mRNAs thatare genuinely associated with the polysomes will fractionate with thepolysomal pellet in the absence of EDTA and with the nonpolysomalsupernatant in the presence of EDTA. The experiment was repeatedonce.

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3�UTR Deletions That Abolish DeadenylationAlso Stabilize the mRNA

The stabilization of endogenous cyclin A1 mRNA in�-amanitin–treated embryos (Fig. 4) may be due to ablock in zygote-mediated deadenylation, or could alsoresult from inhibiting the zygotic expression of a com-ponent necessary to degrade cyclin A1 mRNA. To dis-tinguish between these possibilities, we used chimericmRNAs containing different regions of the cyclin A13�UTR to ask whether deleting a region previouslyshown to be necessary for deadenylation also results inmRNA stabilization.

Figure 1B shows a schematic of chimeric mRNAsused in this study. Radiolabeled chimeric mRNAs con-taining the full-length cyclin A1 3�UTR (GbA1), or thecyclin A1 3�UTR with either the first 309-nt(GbA1�XH) or the first 200-nt (GbA1�XS) deleted wereinjected into two-cell embryos. RNA was extracted andchanges in the stability of the injected mRNA wereassayed by denaturing agarose gel electrophoresis,transfer to membrane, and phosphorimaging of theradioactive signal. RNA loading was verified by prob-ing the membrane for ODC mRNA, and transcriptionwas monitored by probing for GS17 mRNA. Figure 5shows that GbA1 mRNA containing the full-length cy-clin A1 3�UTR is rapidly destabilized after 7 hpf, sim-

ilar to endogenous cyclin A1 mRNA (compare Fig. 5Awith 4). Destabilization occurs after the onset of tran-scription, as seen by the appearance of GS17 mRNA. Incontrast, GbA1�XH and GbA1�XS are present until atleast 15 hpf, well after the initiation of transcription.Quantification of the phosphorimages (Fig. 5B) showsthat the deletion mutants are stabilized 10-fold com-pared with GbA1. These data are consistent with thestabilization of cyclin A1 mRNA in �-amanitin–treatedembryos being due to a block in deadenylation ratherthan to a block in mRNA degradation per se.

The First 200 nt of the Cyclin A1 3�UTR AreSufficient for Deadenylation

Having demonstrated that both endogenous mater-nal cyclin A1 mRNA and chimeric mRNA containingthe cyclin A1 3�UTR are deadenylated by the samemechanism, we next aimed to determine the shortestelement of the cyclin A1 3�UTR sufficient to drive zy-gote-dependent deadenylation. The proximal 200 nt ofthe 3�UTR contains all the sequence information re-quired for zygote-mediated deadenylation (Fig. 1A,bounded by the restriction sites XbaI and SpeI). Achimeric mRNA containing the cyclin A1 3�UTR de-leted of this 200-nt region (GbA1�XS, Fig. 1B) is notdeadenylated after the MBT (Audic et al., 2001), nor is

Fig. 4. Destabilization of cyclin A1 mRNA follows polysome releaseand requires transcription. A: Total RNA was extracted from buffer (Con-trol) and �-amanitin–injected embryos at the indicated times after fertili-zation. Northern analysis was performed with cDNA probes specific forcyclin A1, ornithine decarboxylase (ODC), and GS17. The same blot wasstripped and reprobed successively. hpf, hours postfertilization B: Quan-titation of the RNA present at different times was performed by using aPhosphorImager. Relative RNA levels are shown on the X-axis withmaximal RNA levels equal to 100%. The experiment was repeated twice,and representative results are shown. pf, hours postfertilization

Fig. 5. The 3� untranslated region that is necessary for deadenylationis also required for mRNA destabilization. A: Capped, radiolabeledGbA1, GbA1�XH, and GbA1�XS transcripts were injected into Xenopusembryos. Total RNA was extracted at the indicated times, subjected toMOPS/formaldehyde gel electrophoresis, and transferred to Nytran. In-jected mRNA, phosphorimage of injected mRNA transferred to Nytran.ODC, phosphorimage of Northern analysis with an ornithine decarboxyl-ase cDNA probe. ODC mRNA serves as loading control. GS17, phos-phorimage of Northern analysis with a GS17 specific probe, to show theinitiation of transcription. hpf, hours postfertilization B: Quantitation ofrelative levels of each chimeric mRNA at the times indicated. pf, hourspostfertilization. The experiment was repeated twice, and representativeresults are shown.

515TRANSLATIONAL SILENCING OF CYCLIN A1

it destabilized (Fig. 5). To test whether this 200-ntregion is sufficient for deadenylation, or if additionalsequence/structural information is needed, chimericmRNAs containing either the full-length cyclin A13�UTR (GbA1), the 200-nt region (GbA1�SE), or a309-nt region (GbA1�HE) were injected into embryos.The chimeric mRNAs are shown in Figure 1B. Thesedeletions remove the endogenous CPE and NPS. Thus,the CPE and NPS of the maternal histone B4 mRNA(Paris and Philippe, 1990) were included downstreamof the 3�UTR fragments to ensure that the transcriptswould be adenylated in vivo upon injection. Unadeny-lated, radiolabeled chimeric mRNAs were injected intotwo-cell embryos beginning at 1.5 hpf, and their adeny-lation status was assayed by mobility changes of theextracted RNA on denaturing acrylamide gels.

Figure 6A shows that all chimeric mRNAs are ad-enylated by 3 hpf and remain so until 5 hpf. As ex-pected, deadenylation of GbA1 commences by 7 hpf,shown by the more quickly migrating species and thepresence of fully deadenylated mRNA. By 9 hpf, all ofthe injected GbA1 mRNA is fully deadenylated, migrat-ing at the size of the unadenylated mRNA. GbA1�SEand GbA1�HE likewise decrease in size beginning at 7hpf, with fully deadenylated mRNA present at thistime. Identical to the mRNA containing the wild-type3�UTR, all of the mRNA is fully deadenylated by 9 hpf.Quantification of the phosphorimages in Figure 6B ver-ifies that the kinetics of deadenylation are the same for

all three chimeric mRNAs. We conclude that the prox-imal 200 nt of the 3�UTR are necessary and sufficientfor zygote-mediated deadenylation.

AREs Are Dispensable for Zygote-MediatedDeadenylation

The region of the cyclin A1 3�UTR that is necessaryand sufficient for deadenylation contains two consen-sus class I AREs (Zubiaga et al., 1995) at position 1417(UUAUUUAUU) and position 1491 (UUAUUUAU)(capitalized in Fig. 1A). Because AREs have beenshown to trigger rapid deadenylation of mRNAs in bothXenopus and mammalian cells (Xu et al., 1997; Voeltzand Steitz, 1998), we tested whether these comprisethe zygotic deadenylation element. Mutation of thecentral U to G (AUUUA3AUGUA) has been shown tostabilize AUUUA containing mRNAs (Rajagopalan etal., 1995; Rajagopalan and Malter, 1996). Therefore,we mutated the central U in one or both of the elementsin the context of the chimeric GbA1 mRNA. Radiola-beled GbA1 mRNA, possessing mutations in one orboth of the AU-rich elements, were injected into two-cell embryos. The adenylation status of the injectedmRNAs was assayed by denaturing gel electrophoresisand autoradiography on RNA extracted immediatelyafter injection and then every 2 hr. Results were iden-tical for both of the single mutations and the doublemutation and, thus, are shown only for the doublemutant, GbA1-DM (Fig. 7A).

Figure 7A shows that, as expected, GbA1 was adeny-lated after injection and remained adenylated throughat least 5 hpf. Deadenylation of GbA1 was evident by 7hpf and completed by 9 hpf. Likewise, GbA1-DM isadenylated after injection, deadenylation has begun by7 hr, and the mRNA is completely deadenylated by 9hpf. These results suggest that deadenylation specifiedby the cyclin A1 3�UTR is not ARE-mediated. To ensurethat ARE activity is completely abolished, the experi-ments were repeated after deleting an 81-nt regioncontaining the consensus AREs along with the inter-vening sequence from GbA1�SE (indicated by arrow-heads in Fig. 1A). Figure 7B shows that even in thecomplete absence of AREs (Gb�SE-�ARE), the remain-ing 119 nt of the 3�UTR are sufficient for rapid dead-enylation. The slower kinetics of deadenylation in Fig-ure 7B compared with Figure 7A is due to slowerdevelopment caused by lower ambient temperatureduring the experiment.

To further restrict the deadenylation element, a chi-meric mRNA containing only the first 99 nt of the3�UTR (GbA1�384, Fig. 1B) was assayed for deadeny-lation (Fig. 7C). As expected, the B4 CPE drives arobust polyadenylation of the chimeric mRNA aftermicroinjection. The injected RNA remains polyadenyl-ated until 7 hpf and is then rapidly deadenylated,accumulating as a poly(A)- RNA by 9 hpf. Therefore,the sequence encompassing nt 1318 to 1416 is suffi-cient to confer deadenylation to the chimeric mRNA.Within this region, nt 1371-1386 are capable of forming

Fig. 6. The first 200 nucleotides of the cyclin A1 3� untranslatedregion are sufficient for deadenylation. A: Capped radiolabeled GbA1,GbA1�HE, and GbA1�SE transcripts were injected into Xenopus em-bryos. Total RNA was extracted at the indicated times. The adenylationbehavior of the injected transcripts was analyzed by denaturing gelelectrophoresis and phosphorimaging. The sizes of the RNA molecularweight markers (M) are indicated on the left side of the gels. hpf, hourspostfertilization; U, uninjected transcript.B: Quantification of the percent-age of polyadenylated chimeric transcript (poly(A)�) at each time point.Representative results from one of three experiments are shown. pf,hours postfertilization.

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a stem-loop structure, as predicted by the mfold algo-rithm (Mathews et al., 1999; Zuker et al., 1999) orGenebee RNA2 prediction (Brodsky et al., 1992, 1995).The potential 16-nt stem-loops are shown in Figure 7D.The mfold predicted structure has a 6-bp stem with a4-bp loop and a free energy of �5.8 kcal/mol. TheGenebee predicted structure has a 5-bp stem with a6-bp loop and a free energy of �8.9 kcal/mol.

DISCUSSION

In this report, we show that zygote-mediated dead-enylation of endogenous cyclin A1 mRNA precedes itstranslational repression and destabilization, thereforeprecluding the further accumulation of cyclin A1 pro-tein after the MBT. We also show that the AREspresent in the cyclin A1 3�UTR are dispensable for thiszygote-mediated pathway of mRNA deadenylation. Thecis-acting element sufficient for deadenylation resideswithin the proximal 99 nt of the 3�UTR. This regioncontains a potential 16-bp stem-loop structure (Fig. 7D)

at positions 1371-1386. A single stem loop in the 3�UTRof granulocyte colony-stimulating factor mRNA hasbeen reported recently to promote deadenylation anddestabilization (Putland et al., 2002). Except for thispotential stem-loop structure, no known deadenylationor instability elements are contained in this sequence.Disruption of this sequence will be necessary to ascer-tain whether this is the correct secondary structure,and whether this structure is necessary for deadenyla-tion of cyclin A1. Neither the position of this sequencerelative to the 3� end of the mRNA, nor the length of the3�UTR, affect the deadenylation behavior of the chi-meric mRNA. Chimeric mRNAs with a 3�UTR rangingfrom 483 to 99 nt in length display the same deadeny-lation behavior.

CPEs act as translational repressors in immatureXenopus oocytes and have been shown recently to con-trol cyclin B1 translation both positively and nega-tively in the in vitro embryonic cell cycle (Groisman etal., 2002). CPEB binds to the CPE in the cyclin B1mRNA and interacts with Maskin. Maskin simulta-neously binds the translation initiation factor eIF4E,precluding the binding of eIF4G to eIF4E and inhibit-ing translation (Mendez and Richter, 2001). In each ofour experiments, a CPE was present, either the endog-enous cyclin A1 CPE or the exogenous histone B4 CPE,to obtain in vivo polyadenylation of mRNAs in fertil-ized eggs. Therefore, one could envisage that the CPEis the deadenylation element responsible for cyclin A1deadenylation and translational repression after theMBT. However, this is unlikely as chimeric mRNAscontaining the histone B4 CPE and NPS but not thecyclin A1 3�UTR remain stably polyadenylated until atleast 10 hpf (Audic et al., 1997). It is possible that azygote-transcribed RNA binding protein binds to thedeadenylation element in the cyclin A1 3�UTR andhelps to recruit Maskin, therefore, inhibiting transla-tion. This scenario does not explain the rapid deadeny-lation followed by destabilization. Alternatively, zy-gotic protein binding may destabilize interactionsbetween CPSF, CPEB, and PAB (Dickson et al., 1999),leading to decreased polyadenylation and a concomi-tant increase in deadenylation. Further insight intothe mechanism must await identification of an RNAbinding protein.

Posttranscriptional control is critical in the regula-tion of cyclin levels during the cell cycle. In a humanbreast cancer cell line (Lebwohl et al., 1994), a 3�UTR-truncated cyclin D1 gene encodes a stable cyclin D1mRNA, whereas a small deletion in the 3�UTR wasseen in some leukemias (Hosokawa et al., 1998). Thecyclin D1 3�UTR contains AU-rich sequences and in-teracts with AUF1 (hnRNPD), a protein that binds toAREs and regulates mRNA stability. Cyclins A and B1mRNAs are stabilized in colorectal carcinoma cells bythe ARE-binding protein HuR (Wang et al., 2000). Huproteins were first identified as antigens in lung carci-nomas (Ma et al., 1996) and are also found in Xenopus(Good, 1995). ELAV-related protein A (ElrA) is the

Fig. 7. Cyclin A1 AU-rich elements (AREs) do not target rapid dead-enylation. A: Capped, radiolabeled GbA1 and GbA1DM transcripts wereinjected into Xenopus embryos. GbA1-DM has the central U of each AREmutated to a G. Total RNA was extracted at the indicated times. Theadenylation behavior of the injected transcripts was analyzed by dena-turing gel electrophoresis and phosphorimaging. B: Capped, radiola-beled GbA1�SE and GbA1�SE-�ARE transcripts were injected intoXenopus embryos. GbA1�SE-�ARE has nucleotides (nt) 1417-1498deleted (see Fig. 1B), resulting in the loss of both AREs along with theintervening sequence. C: GbA1�384 (containing only the first 99 nt of thecyclin A1 3� untranslated region or GbA1 transcripts were injected intoembryos and assayed as above. Results are shown for the GbA1�384mRNA. Total RNA was extracted at the indicated times, and the adeny-lation behavior of the injected transcripts was analyzed by denaturing gelelectrophoresis and phosphorimaging. The experiments were repeatedat least three times. D: Cyclin A1 3�UTR stem-loop at nt 1371-1386 aspredicted by the mfold algorithm (Mathews et al., 1999; Zuker et al.,1999) or Genebee RNA2 prediction (Brodsky et al., 1992, 1995). Thearrows in A–C indicate the position of completely unadenylated transcript(Poly A-). hpf, hours postfertilization. U, uninjected transcript.

517TRANSLATIONAL SILENCING OF CYCLIN A1

Xenopus homolog of HuR (Good, 1995). Because theAREs in the cyclin A1 3�UTR are dispensable for dead-enylation at the MBT, AREs and ARE-binding proteinssuch as ElrA do not appear to be involved in its desta-bilization. Destabilization of maternal cyclin A1mRNA, thus, appears to differ from that of somaticcyclin A mRNA.

Replacement of the maternal cyclin A by the somaticcyclin A has been observed in the early development ofboth frogs and mice (Howe et al., 1995; Fuchimoto etal., 2001). The functional significance of this replace-ment is unclear. It has been hypothesized to be in-volved in either the meiotic to mitotic transition(Fuchimoto et al., 2001), or in a switch from maternalto zygotic control of cell cycle progression (Howe et al.,1995). In Xenopus, before the MBT, the embryonic cellcycle is composed of a succession of S and M phases,enabling the cells to divide synchronously every 25–30min. After the MBT (12th cell division), cell divisionslows and becomes asynchronous as gap phases areadded to the cell cycle (Frederick and Andrews, 1994;Newport and Kirschner, 1984). This cell cycle remod-eling is accompanied by alterations in the protein levelof cell cycle regulators, including the disappearance ofcyclins E1, A1, and B2 (Howe et al., 1995; Hartley etal., 1996). Cyclin A1 protein is degraded during eachcell cycle and reaccumulates due to translation duringcell divisions 1–12. Cyclin A1 protein disappears afterdivision 13, and this down-regulation requires zygotictranscription (Audic et al., 2001). We now show thatmaternal cyclin A1 mRNA is released from polysomes(translation ceased) and then degraded during thistime, therefore precluding reaccumulation of cyclinprotein in the subsequent cell cycle. Preceding theseevents, maternal cyclin A1 mRNA is rapidly deadeny-lated by a mechanism requiring zygotic transcriptionand a 99-nt region of the 3�UTR. These data favor amodel in which zygote-driven deadenylation of cyclinA1 mRNA triggers the disappearance of cyclin A1 pro-tein.

It was reported previously that destabilization ofcyclin A1 mRNA in the gastrula stage embryo wasindependent of new transcription (Howe et al., 1995).The discrepancies between the earlier and the currentstudy may result from the use of different inhibitors toblock transcription. The earlier study used the ribonu-cleotide reductase inhibitor, hydroxyurea, which re-duces but does not block transcription and can be toxicto the embryos. The current study used �-amanitin, aspecific inhibitor of RNA polymerase II, and monitoredtranscriptional inhibition by Northern analysis for azygote-specific transcript. In addition, embryos re-mained visually healthy past the usual time of cellcycle remodeling.

The maternal mRNA encoding cyclin B2 is also dead-enylated at the MBT in a zygote-dependent manner. A50-nt region of the 3�UTR is necessary but may not besufficient for this deadenylation (Audic et al., 2001).There is no obvious sequence or structural homology

between this 50-nt region and the 99-nt regulatoryregion of the cyclin A1 3�UTR. In particular, the stem-loop structure identified within the regulatory region ofthe cyclin A1 3�UTR is not detected in the cyclin B23�UTR.

Deadenylation and destabilization is not a defaultstate for cyclin mRNAs, as the mRNA for cyclin B1remains associated with the polysomes (Fig. 3) andcyclin B1 protein persists past the MBT, until at leastlate gastrula stages (Hartley et al., 1996; Audic et al.,2001). Cyclin E1 mRNA is stably polyadenylated in thegastrula (10 hpf) consistent with its continued presence(Audic et al., 2001). Maternal mRNAs encoding cellcycle proteins such as cdc25A (Kim et al., 1999) andXChk1 (Kappas et al., 2000) and other proteins such asc-mos and aurora A (Eg2) are also destabilized after theMBT. c-mos and aurora A mRNA are deadenylatedbefore the MBT in an EDEN-BP–dependent manner(Paillard et al., 1998, 2002) that does not require zy-gotic transcription. The adenylation behavior of cdc25Aand XChk1 has not been studied in Xenopus, thus theyremain potential targets for zygote-mediated deadeny-lation. Consistent with this hypothesis, Drosophilacdc25 mRNA is degraded by a zygotic-mediated path-way (Bashirullah et al., 1999). The zygotic pathway formRNA degradation in Drosophila functions in coordi-nation with the maternal pathway to ensure degrada-tion of specific maternal transcripts by the MBT. Themechanism of zygotic destabilization of maternal tran-scripts in Drosophila is not known.

In conclusion, we have defined a zygotic pathway ofmaternal mRNA destabilization that is dependent on3�UTR regulatory regions that specify deadenylationand translational repression. We have determined thatthe endogenous cyclin A1 mRNA is a substrate of thisdeadenylation pathway. It will now be possible to mu-tate the restricted (99-nt) deadenylation element in thematernal transcript to determine the importance ofthis pathway in establishing the adult cell cycle and inthe transition to zygotic control of development.

EXPERIMENTAL PROCEDURESEmbryos and Microinjections

Xenopus embryos were obtained following standardprotocols (Peng, 1991) and maintained in 0.1� MMR at23°C. For microinjection of mRNA, two-cell embryoswere injected with 18.4 nl of in vitro transcribed,capped mRNA in water (50,000–100,000 cpm/l;0.25–1 fmol/l). �-Amanitin was added (50 ng per em-bryo) where indicated. For adenylation, Northern, andPAT analyses, five embryos were collected at the spec-ified times and processed for RNA extraction as de-scribed (Harland and Misher, 1988) or by following theTriZol protocol (Gibco-BRL). The embryos were stagedaccording to Nieuwkoop and Faber (1967). Initiation oftranscription was monitored by Northern analysis forthe zygote-specific transcript GS17 (Krieg and Melton,1985). Dr. Paul Krieg (University of Arizona, Tucson)generously provided the cDNA for GS17.

518 AUDIC ET AL.

For Northern blot analyses, 10 g of total RNA waselectrophoresed on a MOPS/formaldehyde gel, capil-lary transferred onto Nytran (S&S), and hybridizedwith the indicated cDNA probes. Probes were radiola-beled by random priming by using the Prime-a-Genekit (Promega). Quantitation of phosphorimages wasperformed by using Imagequant software (MolecularDynamics).

Poly(A)Tail Analysis

Total RNA was extracted from embryos injected withradiolabeled, capped chimeric mRNAs and resolved byelectrophoresis on 4% polyacrylamide-urea gels as de-scribed (Audic et al., 2001). Fixed and dried gels wereanalyzed by autoradiography or phosphorimaging (Mo-lecular Dynamics).

The ligation-mediated poly(A) test (LM-PAT) wasperformed as described previously (Salles et al., 1999).Reverse transcription was performed by using oligo-d(T) and an oligo-d(T)-anchor (5�-gcgagctccgcggc-cgcgtttttttttttt). PCR was performed on the resultingcDNA with the oligo-d(T) anchor and a cyclin A13�UTR-specific primer (nt 1318-1335: 5�-agccttccagagt-ggacg). PCR conditions were (94°C, 180 sec; 49°C, 60sec; 72°C, 90 sec) for 1 cycle; (94°C, 60 sec; 49°C, 60 sec;72°C, 90 sec) for 35 cycles; 72°C, 7 min. PCR productswere resolved on a 2% Nusieve (FMC) or low meltingpoint (Gibco-BRL) agarose gel and visualized byethidium bromide staining. Images were reversed tobetter visualize bands. The specificity of PCR amplifi-cation was verified by restriction endonuclease diges-tion using AvaII (not shown).

Polysome Fractionation

Polysomes were isolated from embryos at eitherStage 7 (4 hpf) or Stage 10 (9 hpf) as described (Fritzand Sheets, 2001). Briefly, 20 embryos were homoge-nized on ice in 500 l of polysome buffer (PB). Anadditional 500 l of PB was added, and homogenateswere spun at 12,000 � g 4°C for 15 min. The superna-tant was removed and transferred to a 15-ml falcontube containing either 2 ml of PB or 2 ml of PB plusEDTA, containing 25% (w/v) sucrose and subjected toultracentrifugation at 149,000 � g, 4°C for 2 hr, in aBeckman SW55Ti rotor. The supernatant fractionswere recovered and nonpolysomal RNA isolated withtwo extractions of phenol/chloroform. RNA was iso-lated from the polysomal pellet fractions by TriZol ex-traction. To ensure that RNA pelleting was due to itsassociation with polysomes, each fractionation was re-peated with the addition of EDTA. EDTA chelatesMg2� causing polysomes to dissociate and releasebound mRNAs into the supernatant fraction. Theamount of mRNA in the combined polysome and non-polysome fractions was compared with the amount ofmRNA present in unfractionated material for eachstage of development.

Chimeric Genes and In Vitro Transcription

Cyclin A1 3�UTR with a 5�XbaI and a 3� EcoRV siteadded was generated by PCR as described (Audic et al.,2001). The vector pGbORF/mosEDEN was digestedwith XbaI and EcoRV and the cyclin A1 3�UTR in-serted, replacing the mosEDEN (Fig. 1B) and resultingin the pGbA1 plasmid. pGbA1�HE was constructed bydigesting pGbA1 with HpaI and HindIII. As this dele-tion removed the endogenous CPEs presumably neces-sary for polyadenylation, a dimer of oligonucleotidescomposing the sequence of the histone B4 CPE andNPS was inserted between HpaI and HindIII (B4up15�-taggtttttaatgtttaattctataaataaagtctagatatca; B4dw5�-agcttgatctctagactttatttatagaattaaacattaaaaaccta).Likewise, pGbA1�SE was produced by digesting withSpeI and EcoRV and adding the histone B4 CPE withthe oligonucleotides B4dw and B4up2 (5�-ctagtaggtttt-taatgtttaattctataaataaagtctagatatca). pGbA1�XS wasconstructed by deleting the XbaI/SpeI fragment frompGbA1 and recircularizing the vector. SimilarlypGbA1�XH was generated by deletion of the XbaI/HpaI fragment from pGbA1. The XbaI and HpaI siteswere blunt-ended, and the vector was recircularized.The endogenous CPEs in the 3� half of the 3�UTR isintact in both of these deletion mutants.

The central Us in the AUUUA elements at positions1421 and 1495 were changed to a G by site-directedmutagenesis in the context of pGbA1. These elementsand the intervening sequence (81 nt, 1417-1498) weredeleted from the pGbA1�SE by PCR by using primerstargeted to sequences 5� and 3� of the two AREs. PCRresulted in replication of the entire plasmid minus theAREs and the intervening sequence (pGbA1�SE-�ARE). pGbA1�384 was prepared by digestingpGbA1�SE-�ARE with BamHI, a site that was intro-duced at nt 1416 for preparation of the �ARE con-struct, and SpeI and recircularizing.

All constructions were verified by sequencing. For invitro transcription, all plasmids were linearized withEcoRV. 32P-labeled, capped mRNAs were synthesizedaccording to the manufacturer’s instructions (Strat-agene). A schematic of the resulting chimeric mRNAsis shown in Figure 1B. The pGbORF/mosEDEN plas-mid was generously provided by Dr H.B. Osborne (Uni-versite de Rennes I, France; Paillard et al., 1998).

ACKNOWLEDGMENTS

We thank H.B. Osborne (University of Rennes,France) and P. Krieg (University of Arizona, Tucson)for generously supplying cDNA clones. R.S.H. wasfunded by a grant from the American Cancer Society.M.S. is supported by the Pew Charitable Trust and theJames D. Shaw and Dorothy Shaw Fund of the GreaterMilwaukee Foundation.

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