Cytoplasmic Polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent Mechanisms Regulate Early Class Maternal mRNA Translational Activation in Xenopus Oocytes
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313837200
ISSN1083-351X
AutoresAmanda Charlesworth, Linda L. Cox, Angus M. MacNicol,
Tópico(s)RNA modifications and cancer
ResumoMeiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3′-untranslated region (3′-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3′-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3′-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression. Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3′-untranslated region (3′-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3′-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3′-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression. Early developmental cell fate decisions are regulated through the translational activation of maternally derived mRNAs. Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of mRNAs encoding key regulatory proteins (1Hochegger H. Klotzbucher A. Kirk J. Howell M. le Guellec K. Fletcher K. Duncan T. Sohail M. Hunt T. Development. 2001; 128: 3795-3807Crossref PubMed Google Scholar, 2Nakajo N. Yoshitome S. Iwashita J. Iida M. Uto K. Ueno S. Okamoto K. Sagata N. Genes Dev. 2000; 14: 328-338PubMed Google Scholar, 3Howard E.L. Charlesworth A. Welk J. MacNicol A.M. Mol. Cell Biol. 1999; 19: 1990-1999Crossref PubMed Scopus (74) Google Scholar, 4Sheets M.D. Wu M. Wickens M. Nature. 1995; 374: 511-516Crossref PubMed Scopus (204) Google Scholar, 5Gross S.D. Schwab M.S. Taieb F.E. Lewellyn A.L. Qian Y.W. Maller J.L. Curr. Biol. 2000; 10: 430-438Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 6Ferby I. Blazquez M. Palmer A. Eritja R. Nebreda A.R. Genes Dev. 1999; 13: 2177-2189Crossref PubMed Scopus (149) Google Scholar, 7Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (463) Google Scholar, 8Dupre A. Jessus C. Ozon R. Haccard O. EMBO J. 2002; 21: 4026-4036Crossref PubMed Scopus (91) Google Scholar, 9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar). The translational induction of many maternal mRNAs has been correlated with an increase in mRNA poly(A) tail length. This evolutionarily conserved process, termed cytoplasmic polyadenylation, requires a polyadenylation hexanucleotide sequence (typically AAUAAA) as well as additional 3′-UTR 1The abbreviations used are: 3′-UTR, 3′-untranslated region; CPE, cytoplasmic polyadenylation element; CPEB, cytoplasmic polyadenylation element-binding protein; PRE, polyadenylation response element; GST; glutathione S-transferase; EMSA, electrophoretic mobility shift assay; MAP kinase, mitogen-activated protein kinase; MPF, maturation/M-phase- promoting factor; GVBD, germinal vesicle breakdown; nt, nucleotide; RT, reverse transcriptase; FGF, fibroblast growth factor; FGFR, FGF receptor. regulatory sequences, including cytoplasmic polyadenylation elements (CPE) (10Richter J.D. Sonenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 785-805Google Scholar). CPE sequences have been shown to repress mRNA translation in immature oocytes and to direct cytoplasmic polyadenylation and translational activation in maturing oocytes. Both aspects of CPE function require the CPE-binding protein (CPEB) (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar, 11Fox C.A. Sheets M.D. Wickens M.P. Genes Dev. 1989; 3: 2151-2162Crossref PubMed Scopus (271) Google Scholar, 12McGrew L.L. Dworkin-Rastl E. Dworkin M.B. Richter J.D. Genes Dev. 1989; 3: 803-815Crossref PubMed Scopus (326) Google Scholar, 13McGrew L.L. Richter J.D. EMBO J. 1990; 9: 3743-3751Crossref PubMed Scopus (135) Google Scholar, 14Paris J. Richter J.D. Mol. Cell Biol. 1990; 10: 5634-5645Crossref PubMed Scopus (129) Google Scholar, 15Sallès F.J. Darrow A.L. O'Connell M.L. Strickland S. Genes Dev. 1992; 6: 1202-1212Crossref PubMed Scopus (96) Google Scholar, 16Standart N. Dale M. Dev. Genet. 1993; 14: 492-499Crossref PubMed Scopus (20) Google Scholar, 17Gebauer F. Xu W. Cooper G.M. Richter J.D. EMBO J. 1994; 135712Crossref PubMed Scopus (208) Google Scholar, 18Stebbins-Boaz B. Hake L.E. Richter J.D. EMBO J. 1996; 15: 2582-2592Crossref PubMed Scopus (244) Google Scholar, 19Stutz A. Conne B. Huarte J. Gubler P. Volkel V. Flandin P. Vassalli J.D. Genes Dev. 1998; 12: 2535-2548Crossref PubMed Scopus (99) Google Scholar, 20de Moor C.H. Richter J.D. EMBO J. 1999; 18: 2294-2303Crossref PubMed Scopus (174) Google Scholar, 21Minshall N. Walker J. Dale M. Standart N. RNA (N. Y.). 1999; 5: 27-38Crossref PubMed Scopus (56) Google Scholar, 22Barkoff A.F. Dickson K.S. Gray N.K. Wickens M. Dev. Biol. 2000; 220: 97-109Crossref PubMed Scopus (79) Google Scholar, 23Tay J. Hodgman R. Richter J.D. Dev. Biol. 2000; 221: 1-9Crossref PubMed Scopus (147) Google Scholar). Using progesterone-stimulated oocytes from the frog Xenopus laevis, it has been demonstrated that individual mRNAs are polyadenylated and translationally activated at different times following meiotic cell cycle resumption (24Ballantyne S. Daniel D.L.J. Wickens M. Mol. Biol. Cell. 1997; 8: 1633-1648Crossref PubMed Scopus (88) Google Scholar, 25de Moor C.H. Richter J.D. Mol. Cell Biol. 1997; 17: 6419-6426Crossref PubMed Scopus (131) Google Scholar, 26Sheets M.D. Fox C.A. Hunt T. Vande Woude G. Wickens M. Genes Dev. 1994; 8: 926-938Crossref PubMed Scopus (286) Google Scholar). Because all regulated mRNAs hitherto examined contain CPE sequences, the basis of this temporal order of translational activation was unclear. Various mechanisms been proposed to account for both early and late mRNA cytoplasmic polyadenylation and translational activation profiles during progesterone-stimulated meiotic cell cycle progression. These include the precise nucleotide sequence of the CPE, the number of CPEs, and/or the position of the CPEs within the 3′-UTR (24Ballantyne S. Daniel D.L.J. Wickens M. Mol. Biol. Cell. 1997; 8: 1633-1648Crossref PubMed Scopus (88) Google Scholar, 25de Moor C.H. Richter J.D. Mol. Cell Biol. 1997; 17: 6419-6426Crossref PubMed Scopus (131) Google Scholar, 27Mendez R. Richter J.D. Nat. Rev. Mol. Cell Biol. 2001; 2: 521-529Crossref PubMed Scopus (478) Google Scholar, 28Mendez R. Barnard D. Richter J.D. EMBO J. 2002; 21: 1833-1844Crossref PubMed Scopus (145) Google Scholar). In addition, the cytoplasmic polyadenylation and translational activation of maternal mRNAs has been previously classified as being independent of (class I), or dependent on (class II), translation of the mRNA encoding the Mos proto-oncogene (24Ballantyne S. Daniel D.L.J. Wickens M. Mol. Biol. Cell. 1997; 8: 1633-1648Crossref PubMed Scopus (88) Google Scholar, 25de Moor C.H. Richter J.D. Mol. Cell Biol. 1997; 17: 6419-6426Crossref PubMed Scopus (131) Google Scholar). A generalized understanding of the molecular mechanisms governing the temporal control of maternal mRNA translational activation has been confounded by a number of inconsistencies in assignment of CPE-containing mRNAs to particular classes, as well as differences in temporal regulation within class groups. Furthermore, the conversion of CPEB from a translational repressor to a translational activator appears to occur too late to regulate early mRNA cytoplasmic polyadenylation and translational activation. CPEB conversion involves both Aurora A/Eg2- and cdc2-dependent phosphorylation (28Mendez R. Barnard D. Richter J.D. EMBO J. 2002; 21: 1833-1844Crossref PubMed Scopus (145) Google Scholar, 29Mendez R. Hake L.E. Andresson T. Littlepage L.E. Ruderman J.V. Richter J.D. Nature. 2000; 404: 302-307Crossref PubMed Scopus (294) Google Scholar). Cdc2 activation occurs subsequent to the translational activation of the temporally early Mos mRNA (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar), and although initial studies suggested Aurora A/Eg2 activation occurred early during maturation (29Mendez R. Hake L.E. Andresson T. Littlepage L.E. Ruderman J.V. Richter J.D. Nature. 2000; 404: 302-307Crossref PubMed Scopus (294) Google Scholar, 31Andrèsson T. Ruderman J.V. EMBO J. 1998; 17: 5627-5637Crossref PubMed Scopus (122) Google Scholar), subsequent studies (32Maton G. Thibier C. Castro A. Lorca T. Prigent C. Jessus C. J. Biol. Chem. 2003; 278: 21439-21449Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 33Frank-Vaillant M. Haccard O. Thibier C. Ozon R. Arlot-Bonnemains Y. Prigent C. Jessus C. J. Cell Sci. 2000; 113: 1127-1138Crossref PubMed Google Scholar, 34Castro A. Mandart E. Lorca T. Galas S. J. Biol. Chem. 2003; 278: 2236-2241Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) have concluded that Eg2 activation is temporally late and perhaps dependent on prior cdc2 activation. The recent identification of a CPE- and CPEB-independent regulatory mechanism that controls the early translational activation of the Mos mRNA (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar) suggests that CPE- and CPEB-independent regulatory mechanisms may be more generally utilized to generate the range of temporal profiles of maternal mRNA translational activation observed during meiotic cell cycle progression. The limited number of endogenous mRNAs or reporter RNAs that have been studied simultaneously has hampered elucidation of the mechanisms controlling the timing of maternal mRNA translational activation. Moreover, many of the 3′-UTR reporter constructs used previously were not full length thus compromising the generality of the conclusions that could be drawn. To circumvent these prior problems, we have utilized RNA ligation-coupled PCR (35Rassa J.C. Wilson G.M. Brewer G.A. Parks G.D. Virology. 2000; 274: 438-449Crossref PubMed Scopus (62) Google Scholar) from the same oocyte samples to investigate the temporal kinetics of cytoplasmic polyadenylation of multiple endogenous mRNAs during oocyte meiotic maturation. We report that contrary to current dogma, CPE- and CPEB-dependent mechanisms do not control temporally early maternal mRNA polyadenylation and translational activation during Xenopus oocyte maturation. We define a new class of Xenopus maternal mRNAs, which undergo temporally early CPE- and CPEB-independent cytoplasmic polyadenylation and translational activation. We demonstrate that polyadenylation response elements (PREs) present in the 3′-UTRs of early class mRNAs function to direct temporally early cytoplasmic polyadenylation and translational activation. By contrast, the cytoplasmic polyadenylation of CPE- and CPEB-dependent mRNAs is a temporally late event. Our findings suggest that the sequential action of distinct 3′-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression. DNA Constructs—The 211-nt D7 3′-UTR, the 146-nt G10 3′-UTR, and the 71-nt histone-like B4 3′-UTR were isolated from immature Xenopus oocytes by RT-PCR using primers containing 5′ XbaI sites and 3′ SalI sites and cloned into pGEM GST (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar) for polyadenylation and translation studies, or into pGEM4Z (Promega) for RNA EMSAs. It should be noted that the D7 3′-UTR was larger than predicted and was found to encode an additional 21 nucleotides of 3′ sequence not present in the GenBank™ entry, including a canonical AAUAAA polyadenylation hexanucleotide. The histone-like B4 CPE was changed from uuuuuaau to uuuuGGau; the D7 CPEs were changed from uuuuuuaa, uuuuaaca, uuuuuaca, and uuuuuau to uuuuuGGa, uuuGGaca, uuuuGGca, and uuuuGGu; and the G10 CPEs were changed from uuuuaau and uuuuuuau to uuuGGau and uuuuuGGu using QuikChange (Stratagene). The D7 UTR was truncated using standard PCR mutagenesis to remove the two 5′-most CPEs, while leaving the PRE intact (see Fig. 7A for PRE boundary). The PRE in the D7 UTR was deleted by using QuikChange. The B4, D7, and G10 3′-UTR constructs were linearized with SalI prior to in vitro transcription. QuikChange was used to replace β-globin sequence with the histone-like B4 and D7 PREs in pGEM GST β-globin UTR (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar), at the respective distance from the polyadenylation hexanucleotide as they are found in their native mRNAs. Because the D7 PRE spans a CPE, the CPE was disrupted as described above to eliminate any possible contribution to cytoplasmic polyadenylation. pGEM GST CPEB-AA construction and RNA transcription have been described before (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar). pGEM GST XeCPEB has been described (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar). Oocyte Isolation, Culture, Microinjection, and Lysate Preparation— Xenopus oocyte isolation and culture has been described previously (36Machaca K. Haun S. J. Cell Biol. 2002; 156: 75-85Crossref PubMed Scopus (81) Google Scholar). Oocytes were induced to mature with 2 μg/ml progesterone, and pools of 5 or 6 oocytes were taken at the indicated times. Where synthetic RNA constructs were injected, collection of immature controls was performed in concert with time-matched progesterone-stimulated samples. Germinal vesicle breakdown (GVBD) was used as an indicator of maturation and assessed by the presence of a white spot on the animal pole. Where indicated, oocytes were segregated at the time when 50% of the oocyte population had reached GVBD (GVBD50) based on whether they had (+) or had not (-) completed GVBD. RNA was transcribed in vitro with SP6 and injected at concentrations described previously (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar). RNA and protein were extracted from the same pool of oocytes (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar). Total RNA was isolated using RNA STAT-60 (Tel-Test) (3Howard E.L. Charlesworth A. Welk J. MacNicol A.M. Mol. Cell Biol. 1999; 19: 1990-1999Crossref PubMed Scopus (74) Google Scholar). Polyadenylation Assays—RNA ligation-coupled RT-PCR (see Fig. 1A) was a modified version of the technique described previously (35Rassa J.C. Wilson G.M. Brewer G.A. Parks G.D. Virology. 2000; 274: 438-449Crossref PubMed Scopus (62) Google Scholar). 4 μg of total oocyte RNA, from pools of 5 or 6 oocytes, was ligated to 0.4 μg of P1 anchor primer (35Rassa J.C. Wilson G.M. Brewer G.A. Parks G.D. Virology. 2000; 274: 438-449Crossref PubMed Scopus (62) Google Scholar), in a 10-μl reaction using T4 RNA ligase (New England Biolabs) according to the manufacturer's directions. The whole 10-μl RNA ligation reaction was used in a 50-μl reverse transcription reaction using Superscript III (Invitrogen), according to manufacturer's directions using 0.4 μg of P1′ as the reverse primer (35Rassa J.C. Wilson G.M. Brewer G.A. Parks G.D. Virology. 2000; 274: 438-449Crossref PubMed Scopus (62) Google Scholar). 1 μl of this cDNA preparation was used in each 50-μl PCR using Platinum Taq (Invitrogen), according to the manufacturer's directions. PCR was performed for 40 cycles, using a 56 °C annealing temperature and 1.5 mm final concentration of Mg2+, except the FGFR1 reaction which required 2.5 mm Mg2+. The specific primers used were designed to be 70–110 nucleotides from the poly(A) addition site according to sequence information available in GenBank™, and the accession numbers are as follows: X13855 (B4 mRNA for histone H1-like protein); XLD7 (maternal mRNA D7); Z17206 (Aurora A/Eg2); U24491 (FGF receptor 1); XLG10 (maternal G10 mRNA); X13311 (p39mos); J03166 (cyclin B1); AJ304991 (cyclin B4); U13962 (Xe-Wee1A); X53745 (cyclin A1); and M24769 (cytoskeletal actin type 5). The primers used are as follows: histone-like B4, AGT GAC AAA CTA GGC TGA TAT ACT; cyclin A1, CAT TGA ACT GCT TCA TTT TCC CAG; cyclin B1, GTG GCA TTC CAA TTG TGT ATT GTT; cyclin B4, CAT AGG ACA CTT GTT ATA TTG TAG; D7, TGT TGT GAA GTT GCC ATT TAG TAT; Aurora A/Eg2, GTT TCA ATC TTG TAT GTC CTT TTA; FGFR1, TTT GCT ATG TTT TCA GTT TGT ATT; G10, TAA GGC CGG CGA CTG AAA TTG TGT; Mos, GTT GCA TTG CTG TTT AAG TGG TAA; and Wee1, GGC CTG GAC AAA AAC TTT ATA ATT. To verify that an increase in PCR product size in stimulated oocytes is indicative of extension of the poly(A) tail, treatment of the initial RNA samples with oligo(dT) and RNase H (to degrade all poly(A) tails (3Howard E.L. Charlesworth A. Welk J. MacNicol A.M. Mol. Cell Biol. 1999; 19: 1990-1999Crossref PubMed Scopus (74) Google Scholar) prior to RNA ligation with the P1 DNA oligonucleotide) did reduce the size of the PCR product in progesterone-treated samples to a size comparable with that from immature oocyte samples (data not shown). Direct sequencing of representative PCR products also confirmed the increase in PCR product size observed in progesterone-stimulated oocytes was due to poly(A) tail extension. RNA Electrophoretic Mobility Shift Assays—The amount of GST and GST-XeCPEB protein expressed in rabbit reticulocyte lysates was normalized prior to use, and the RNA EMSAs were performed as described previously (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar). The histone-like B4 and truncated D7 probes were run on 3.75% acrylamide gels, the G10 on a 3.5% gel, and the full-length D7 probe on a 3.25% gel. GST Reporter mRNA Translation Assays—To measure the effect of wild-type and mutant 3′-UTRs on GST reporter mRNA translation, GST accumulation was visualized and quantitated by ECL Western blotting as described previously (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar, 30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar) using ChemiGlow West, a ChemiImager 5500 and AlphaEaseFC software (AlphaInnotech Corp.). To address the role of CPEs and CPEB in the control of maternal mRNA cytoplasmic polyadenylation and translational activation, we performed a comparative study of the polyadenylation of multiple endogenous mRNAs from the same sample sets utilizing an RNA ligation-coupled PCR technique (35Rassa J.C. Wilson G.M. Brewer G.A. Parks G.D. Virology. 2000; 274: 438-449Crossref PubMed Scopus (62) Google Scholar). Briefly, a DNA oligonucleotide (P1) is ligated directly onto the 3′ end of all the RNAs in the sample preparation (Fig. 1A, step 1). An antisense DNA oligonucleotide (P1′), complementary to the RNA-ligated P1 oligonucleotide, is then used as a primer for reverse transcription (Fig. 1A, step 2). From this pool of cDNAs, the polyadenylation status of individual mRNAs was assessed by PCR using a gene-specific forward primer and the P1′ reverse primer (Fig. 1A, step 3) enabling a large number of endogenous mRNAs to be analyzed from the same sample preparation. Because we have shown previously that the Mos mRNA undergoes temporally early CPE- and CPEB-independent translational activation (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar), we wished to determine which Xenopus maternal mRNAs undergo similar temporally early cytoplasmic polyadenylation in progesterone-stimulated oocytes. 11 maternal mRNAs were analyzed utilizing the RNA ligation-coupled PCR technique from the same total RNA sample preparations (Fig. 1B). We identify two classes of maternal mRNAs based on the timing of their induced polyadenylation profiles, designated herein as early (where polyadenylation is initiated 2–3 h prior to oocyte germinal vesicle breakdown (GVBD), a marker of meiotic cell cycle progression) and late (where polyadenylation is initiated either 1 h before, or coincident with, GVBD). The early class of mRNAs include histone-like B4, D7, Eg2, FGFR1, G10, and Mos and consistently undergo coordinated initiation of polyadenylation 2–3 h prior to GVBD in oocytes from all animals tested. The polyadenylation of these mRNA populations occurs in a stepwise fashion up until GVBD, at which time the mRNA populations achieve maximal poly(A) tail length. The late class of mRNAs include cyclin B1, cyclin B4, Wee1, and cyclin A1. The mRNA populations of the late class mRNAs behave heterogeneously in terms of when polyadenylation is initiated for oocytes from any given animal. Generally, late class polyadenylation occurred coincident with or after GVBD. However, in some animals (as represented in Fig. 1B) some late class mRNA polyadenylation can be observed up to 1 h prior to GVBD, although this initiation occurred later than that seen with the early class mRNAs, and the entire population of each late class mRNA does not become fully polyadenylated until GVBD. Indeed, whereas cyclin A1 polyadenylation occurs predominantly after GVBD (Fig. 1B, open triangle), a proportion of the cyclin B1, B4, and Wee1 mRNA populations were polyadenylated an hour prior to GVBD (Fig. 1B, open boxes). In all animals examined so far, initiation of cyclin A1 polyadenylation was always the last to occur. The cytoskeletal actin mRNA, which lacks CPE sequences in the 3′-UTR, is utilized as a control (Fig. 1B, actin type 5) because it is not polyadenylated in response to progesterone stimulation and is deadenylated at GVBD (26Sheets M.D. Fox C.A. Hunt T. Vande Woude G. Wickens M. Genes Dev. 1994; 8: 926-938Crossref PubMed Scopus (286) Google Scholar). The timing of maternal mRNA polyadenylation characterized here generally agrees with previous studies that have reported that histone-like B4, G10, D7, FGF receptor 1, and Mos are early (11Fox C.A. Sheets M.D. Wickens M.P. Genes Dev. 1989; 3: 2151-2162Crossref PubMed Scopus (271) Google Scholar, 13McGrew L.L. Richter J.D. EMBO J. 1990; 9: 3743-3751Crossref PubMed Scopus (135) Google Scholar, 24Ballantyne S. Daniel D.L.J. Wickens M. Mol. Biol. Cell. 1997; 8: 1633-1648Crossref PubMed Scopus (88) Google Scholar, 25de Moor C.H. Richter J.D. Mol. Cell Biol. 1997; 17: 6419-6426Crossref PubMed Scopus (131) Google Scholar, 26Sheets M.D. Fox C.A. Hunt T. Vande Woude G. Wickens M. Genes Dev. 1994; 8: 926-938Crossref PubMed Scopus (286) Google Scholar, 30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar, 37Paris J. Philippe M. Dev. Biol. 1990; 140: 221-224Crossref PubMed Scopus (114) Google Scholar, 38Culp P.A. Musci T.J. Dev. Biol. 1998; 193: 63-76Crossref PubMed Scopus (15) Google Scholar), whereas cyclin A1, cyclin B1, and Wee1 are temporally late (9Charlesworth A. Welk J. MacNicol A. Dev. Biol. 2000; 227: 706-719Crossref PubMed Scopus (50) Google Scholar, 24Ballantyne S. Daniel D.L.J. Wickens M. Mol. Biol. Cell. 1997; 8: 1633-1648Crossref PubMed Scopus (88) Google Scholar, 25de Moor C.H. Richter J.D. Mol. Cell Biol. 1997; 17: 6419-6426Crossref PubMed Scopus (131) Google Scholar, 26Sheets M.D. Fox C.A. Hunt T. Vande Woude G. Wickens M. Genes Dev. 1994; 8: 926-938Crossref PubMed Scopus (286) Google Scholar). However, this is the first time it has been shown that the early endogenous mRNAs undergo coordinate initiation. The timing of cyclin B4 and Aurora A/Eg2 mRNA cytoplasmic polyadenylation has not been established previously. Because the temporally early polyadenylation of the Mos mRNA occurs in a CPEB-independent manner (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar), we next wished to determine whether Aurora A/Eg2-mediated CPEB phosphorylation was necessary for the progesterone-stimulated cytoplasmic polyadenylation of the other members of the temporally early class of maternal mRNAs. To this end we employed the expression of a dominant negative form of CPEB (CPEB-AA) (29Mendez R. Hake L.E. Andresson T. Littlepage L.E. Ruderman J.V. Richter J.D. Nature. 2000; 404: 302-307Crossref PubMed Scopus (294) Google Scholar) that has alanine residue substitutions in the Aurora A/Eg2 consensus phosphorylation sites. We demonstrate that in addition to the Mos mRNA, the mRNAs encoding histone-like B4, D7, Aurora A/Eg2, FGFR1, and G10 undergo progesterone-stimulated polyadenylation in the presence of CPEB-AA. It should be noted, however, that the expression of CPEB-AA reduced the overall length of progesterone-stimulated poly(A) tail extension (Fig. 2). Because the reduced poly(A) tail still allows for PRE-directed translational activation of the Mos mRNA in CPEB-AA expressing oocytes (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar), it is likely that the reduced poly(A) tail added to the histone-like B4, D7, Aurora A/Eg2, FGF receptor 1, and G10 mRNAs is sufficient for translational activation. By contrast, polyadenylation of the cyclin B1 (30Charlesworth A. Ridge J.A. King L.A. MacNicol M.C. MacNicol A.M. EMBO J. 2002; 21: 2798-2806Crossref PubMed Scopus (56) Google Scholar), cyclin B4, cyclin A1, and Wee1 mRNAs was completely abolished in CPEB-AA expressing oocytes (Fig. 2). These results demonstrate that there are distinct CPEB-independent and CPEB-dependent classes of Xenopus maternal mRNAs. The CPEB-independent class corresponds to the temporally early mRNAs, whereas the CPEB-dependent class correlates with the temporally late mRNAs shown in Fig. 1. As noted in the Introduction, the sequence, number, and/or position of CPEs have been proposed previously to encode the temporal polyadenylation characteristics of maternal mRNAs. Although there is some variability in precise sequence, Xenopus CPE sequences generally conform to the consensus U4–5A1–3U and function up to 100 nucleotides 5′ of the polyadenylation hexanucleotide (10Richter J.D. Sonenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 785-805Google Scholar, 39Richter J.D. Microbiol. Mol. Biol. Rev. 1999; 63: 446-456Crossref PubMed Google Scholar). A terminal U is not always required for CPE function because UUUUAACA has been implicated in cyclin B1 repression (22Barkoff A.F. Dickson K.S. Gray N.K. Wickens M. Dev. Biol. 2000; 220: 97-109Crossref PubMed Scopus (79) Google Scholar). We find no apparent correlation between temporally early or late class mRNAs and a specific CPE sequence. Indeed, the different CPE sequence permutations (e.g. UUUUAU, UUUUUAAU, and UUUUAACA) can be found in both early and late class mRNAs analyzed in this study (Fig. 3). We also found no general correlation between temporally early or late class mRNAs and CPE number because both classes contain mRNAs with one or more CPEs (Fig. 3). However, CPE position did appear to correlate with the classification of temporally late mRNAs analyzed because they all contain a CPE in the 3′-UTR that overlaps the polyadenylation hexanucleotide. Because the temporally early mRNAs examined herein do contain one or more CPE sequences but none of the CPEs overlap the polyadenylation hexanucleotide, we next wanted to test whether CPE sequences 5′ of the polyadenylation hexanucleotide were responsible for the cytoplasmic polyadenylation of the early class of mRNAs. The full-length 3′-UTRs of histone-like B4, D7, and G10 mRNAs were isolated from Xenopus oocytes, and the consensus CPE sequences in each 3′-UTR were mutationally disrupted with dinucleotide substitutions (Fig. 4A). To verify that the mutations had indeed disrupted the CPEs, the binding to CPEB in vit
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