1 Introduction [1–6] [7] Xenopus [8] [9] Xenopus 2 mRNA elements and RNA binding proteins Xenopus Xenopus [10,11] [10,12–14] [15,16] [17] [18–20] 5 4 4–5 2 [5,6,21,22] Xenopus 5 [23,24] 5 1–2 [21,25–29] [21,30,31] [20,21,31,32] [20,21,31,32] [33,34] [5,6,11,22,28] [35,36] [37] [38–41] [42–45] [46] [40,46] Xenopus [47] [47] [47,48] [46,49] [48] 3 Symplekin [50–52] [53–55] [50,56] Xenopus [50] [57] [57] 4 The cytoplasmic poly(A) polymerase In vitro [47–49] Xenopus [58,59] [58] [60] Caenorhabditis elegans gld-2 [61] [62,63] Xenopus [57] [57] [30] Xenopus Drosophila [64] [65,66] Xenopus [67] [57] [68,69] [65,70] [65] [68,69] 5 The activation of cytoplasmic polyadenylation Xenopus [71–73] Xenopus [62] [57] [57] [74] [74] [74] [74] Xenopus [16,75,76] [15,17] [15,77,78] [15,17,75,78] [15,17,48] [17,32] [21] [15,16,32,79,80] Xenopus [17,81] [32,48,82] [74] [15,32,83] [32] [32] in vitro [48] [83] Xenopus [83] [79,84–86] [79] [87] Xenopus [88–91] [92] [16,17,79,93] [32,79,94] [79] [94] [65,95–98] [80] [80,99] [99] [80,99] [80] [80,99] [99] [79] [79] [100] Xenopus [100] [100] [101] [15,77,78] [16,77,78,93] [101–103] [57] [101] Figs. 1 and 2 1. The activation of CPE-mediated cytoplasmic polyadenylation during progesterone induced oocyte maturation is induced by a drop in protein kinase A activity and requires an early translation event, perhaps translation of RINGO/Speedy. 2. This induces the early activation of MAP kinase, which is associated with the polyadenylation complex through XGef and phosphorylates CPEB on multiple sites, but not on Ser174. 3. CPEB is phosphorylated on Ser174 by an as yet not fully confirmed kinase, possibly Aurora A or CamKII, which is required for the induction of CPE-mediated polyadenylation and causes an increase in the binding between Gld-2, CPSF and CPEB, causing the ejection of PARN from the complex and allowing Gld-2 to elongate the poly(A) tail of the mRNA. 4. After GVBD, CPEB is phosphorylated by cdk1 and the free CPEB is mostly degraded, allowing the CPEB in polyadenylation complexes (now probably phosphorylated on Ser174 by Aurora A) to activate cytoplasmic polyadenylation on mRNAs which contain a CPE overlapping with the poly(A) signal. [16,75,76] [71] [19,26] [104] [105] 6 Deadenylation and translational repression [74,106] [74,106] [107,108] [97,107–109] [110,111] [112–114] [113] [107,115] [116] Xenopus [19,117,118] [119] [105,119–121] [122] Xenopus Xenopus Xenopus [78,123,124] [71] [75,105,107–109,125–127] Fig. 3 [128] [129–134] [129,135–137] [138] [139] [140–142] [74] [74,128] [120] [143] [144] [145] Xenopus [146–148] [149] [113] [143] [67,143] At present it is difficult to choose amongst the multitude of models for translational repression by CPEs. Some of the complexes detected by pulldown and immunoprecipitation may not contain the majority of the repressed mRNA, even though presence of some mRNA was demonstrated by RT-PCR. Alternatively, it may well be that every one of these models is correct at a particular stage of oogenesis or embryogenesis or that the repression is different for specific mRNAs, depending on binding sites for other proteins such as Pumilio and EDEN-BP. An attractive option is that sequestration to P-body like large complexes is the result of translational repression and acts as an enhancer of translational repression, while smaller, more mRNA specific translational repression complexes are formed during the movement of mRNAs in and out of P-bodies. To distinguish between these models, it will be important to characterise the cap binding proteins and P-body components present on specific mRNAs at different developmental stages. 7 Cytoplasmic polyadenylation and translational activation [150] Xenopus [29,74] [6,13] [76] 8 Discussion Xenopus 1. A more systematic investigation of the consensus CPE sequence and its maximum distance to the poly(A) signal would enable a more reliable bioinformatic prediction of the targets of CPE-mediated cytoplasmic polyadenylation. Are all mRNAs containing these sequences polyadenylated during oocyte maturation? 2. The relative roles of Gld-2 and PAP in cytoplasmic polyadenylation need to be further evaluated. Can they substitute for each other? What are their contact points in the cytoplasmic polyadenylation complex? A cytoplasmic polyadenylation system reconstituted from pure components would be ideal to resolve these questions. 3. The kinase(s) responsible for the early activating phosphorylation on Ser174 of CPEB should be identified unequivocally. If Aurora A is responsible, why do inhibitors of its activity not block oocyte maturation and can no early activation of this kinase be detected? Do inhibitors of CamKII affect early polyadenylation? What is the signal transduction cascade leading to induction of the activating kinase? 4. What are the relative roles of Musashi and CPEB in the polyadenylation of c-Mos mRNA and other substrates? How do dominant negative mutants of CPEB and Musashi achieve their repressive functions? May there be off target effects through titration of common polyadenylation factors or signal transduction machinery? 5. in vitro 6. Are the complexes on cytoplasmic polyadenylation sequences other than the CPE similar to the CPEB associated complexes? For instance, do Gld2 or PAP associate with the RNA binding proteins that recognise them? 7. Which models for translational repression and deadenylation of CPE containing mRNAs are correct in what stage of oogenesis? Again, a more intimate knowledge of the direct interactions involved in assembling the repression complexes is likely to yield important new investigative tools for resolving this question. In addition, the study of mRNP complexes assembled in vivo could yield some more conclusive answers. 8. Are the polyadenylation and translation repression complexes identical for each CPE containing mRNA or are there mRNA specific differences in the complexes? Immunoprecipitation followed by RT-PCR and RNA affinity chromatography can partially answer this question, but affinity purification methods that target specific mRNPs are likely to be crucial to obtain a full answer. Xenopus [9,151–153] [9]