Embryo deadenylation element-dependent deadenylation is enhanced by a cis element containing AUU repeats.

The deadenylation of maternal mRNAs in the Xenopus embryo is a sequence-specific process. One cis element that targets maternal mRNAs for deadenylation after fertilization is the embryo deadenylation element (EDEN). This element, composed of U/R repeats, is specifically bound by a protein, EDEN-BP. In the present study we show that the rate at which an RNA containing an EDEN is deadenylated can be increased by the presence of an additional cis element composed of three AUU repeats. This effect was observed for a natural EDEN (c-mos) and two synthetic EDENs. Hence, the enhancement of EDEN-dependent deadenylation conferred by the (AUU)3 motif is not due to an interaction with a particular EDEN sequence. Mutation of the (AUU)3 motif abrogated the enhancement of EDEN-dependent deadenylation. These data indicate that the rate at which a specific maternal mRNA is deadenylated in Xenopus embryos is probably defined by a cross talk between multiple cis elements.

EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos.

During Xenopus early development, gene expression is regulated mainly at the translational level by the length of the poly(A) tail of mRNAs. The Eg family and c-mos maternal mRNAs are deadenylated rapidly and translationally repressed after fertilization. Here, we characterize a short sequence element (EDEN) responsible for the rapid deadenylation of Eg5 mRNA. Determining the core EDEN sequence permitted us to localize the c-mos EDEN sequence. The c-mos EDEN confered a rapid deadenylation to a reporter gene. The EDEN-specific RNA-binding protein (EDEN-BP) was purified and a cDNA obtained. EDEN-BP is highly homologous to a human protein possibly involved in myotonic dystrophy. Immunodepleting EDEN-BP from an egg extract totally abolished the EDEN-mediated deadenylation activity, but did not affect the default deadenylation activity. Therefore, EDEN-BP constitutes the first trans-acting factor for which an essential role in the specificity of mRNA deadenylation has been directly demonstrated.

Postfertilization deadenylation of mRNAs in Xenopus laevis embryos is sufficient to cause their degradation at the blastula stage.

Although the maternal Xenopus laevis Eg mRNAs are deadenylated after fertilization, they are not immediately degraded and they persist in the embryos as poly(A)- transcripts. The degradation of these RNAs is not detected until the blastula stage of development (6 to 7 h postfertilization). To understand the basis for this delay between deadenylation and degradation, it is necessary to identify the cis-acting element(s) required to trigger degradation in blastula stage embryos. To this end, several chimeric RNAs containing different portions of the 3' untranslated region of Eg2 mRNA were injected into two-cell X. laevis embryos. We observed that only the RNAs that contained the cis-acting elements that confer rapid deadenylation were subsequently degraded at the blastula stage. This suggested that deadenylation may be sufficient to trigger degradation. By injecting chimeric RNAs devoid of Eg sequence information, we further showed that only deadenylated RNAs were degraded in X. laevis embryos. Last, introduction of a functional cytoplasmic polyadenylation element into a poly(A)- RNA, thereby causing its polyadenylation after injection into embryos, protected the RNA from degradation. Hence, in X. laevis embryos, the postfertilization deadenylation of maternal Eg mRNAs is sufficient to cause the degradation of an mRNA, which, however, only becomes apparent at the blastula stage. Possible causes for this delay between deadenylation and degradation are discussed in the light of these results.

Substrate-specific regulation of RNA deadenylation in Xenopus embryo and activated egg extracts.

The poly(A) tail of mRNAs plays an important role in translational control. In Xenopus laevis matured oocytes, maternal mRNAs that contain a cytoplasmic polyadenylation element (CPE) are polyadenylated, whereas CPE deficient mRNAs are deadenylated by a default process. Eg mRNAs are maternal transcripts that are poly(A)+ in matured oocytes and rapidly deadenylated after fertilization. This post-fertilization deadenylation of Eg mRNAs requires specific sequence information. Such a deadenylation element has been identified previously in the 3'UTR of Eg2 mRNA. In this study, we show that cell-free extracts made from embryos or activated eggs contain two kinetically distinct deadenylation activities, with different substrate specificities. One, responsible for the slow deadenylation of RNAs that are devoid of a functional CPE, has the characteristics of a default PAN activity. The other effectuates the rapid deadenylation of RNAs containing a deadenylation element. The in vitro system described here will allow the characterization of factors controlling the deadenylation of Eg mRNAs in embryos.

The deadenylation conferred by the 3' untranslated region of a developmentally controlled mRNA in Xenopus embryos is switched to polyadenylation by deletion of a short sequence element.

The maternal Xenopus Eg mRNAs are adenylated and translated in the mature oocyte and then, after fertilization, are deadenylated and released from polysomes. Therefore, after fertilization, a change occurs in the cellular mechanisms that control mRNA adenylation. In the study reported here, we show that the 3' untranslated region of Eg2 mRNA contains a cis-acting element that is required for the deadenylation of chimeric RNAs after fertilization. This cis-acting element is contained within a single 17-nucleotide portion of the Eg2 mRNA. Disruption of this deadenylation element allows adenylation of the chimeric transcripts in the embryo. Therefore, this cis-acting element is part of the sequence information required for the developmental switch from adenylation to deadenylation of the maternal Eg2 mRNA in Xenopus embryos.

Identification of RNA-binding proteins specific to Xenopus Eg maternal mRNAs: association with the portion of Eg2 mRNA that promotes deadenylation in embryos.

Maternal Xenopus Eg mRNAs have been previously identified as transcripts that are specifically deadenylated after fertilization and degraded after the mid blastula transition. Destabilizing cis sequences were previously localised in the 3' untranslated region of Eg2 mRNA. In order to characterize possible trans-acting factors which are involved in the post-transcriptional regulation of Eg mRNAs, gel-shift and u.v. cross-linking experiments were performed, which allowed the identification of a p53-p55 RNA-binding protein doublet specific for the 3' untranslated regions of Eg mRNAs. These p53-p55 proteins do not bind to the 3' untranslated regions of either ornithine decarboxylase or phosphatase 2Ac mRNAs, which remain polyadenylated in embryos. These novel RNA-binding proteins are distinct from the cytoplasmic polyadenylation element-binding protein that controls the polyadenylation of maternal mRNAs in maturing Xenopus oocytes, and from previously identified thermoresistant RNA-binding proteins present in oocyte mRNP storage particles. The p53-p55 bind a portion of the Eg2 mRNA 3' untranslated region, distinct from the previously identified destabilizing region, that is able to confer the postfertilization deadenylation of CAT-coding chimeric mRNAs. This suggests that the p53-p55 RNA-binding proteins are good candidates for trans-acting factors involved in the deadenylation of Eg mRNAs in Xenopus embryos.

Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements.

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.

Characterization of SV40 enhancer motifs involved in positive and negative regulation of the constitutive late promoter activity; effect of T-antigen.

By analyzing the late promoter activity of a series of nonreplicative recombinants mutated within the different enhancer motifs of SV40 we identified both positive and negative regulatory elements. In the absence of T-antigen, the motifs Sph and/or octamer, and to a lesser extent the motifs GTI and P, account for the constitutive expression of the late promoter. The motif GTII overlaps elements that negatively regulate the expression of the late promoter. These results indicate that the late promoter is down-regulated not only at the level of the GC motifs but also at the enhancer level. Moreover, we showed that T-antigen interacts with both positive and negative regulatory elements.

Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2.

Fertilization of Xenopus laevis eggs triggers a period of rapid cell division comprising 12 nearly synchronous mitoses. Protein synthesis is required for these divisions, and new proteins appear after fertilization. Others proteins however, which are synthesized in the unfertilized egg, are no longer made in the early embryo. To identify such proteins, a differential screen of an egg cDNA library gave nine clones corresponding to mRNAs that are deadenylylated soon after fertilization. The sequence of one of these clones (Eg1) revealed a high homology to p34cdc2, the kinase subunit of maturation-promoting factor. Only 12 amino acids in the deduced amino acid sequence were unique to Eg1 when its sequence was compared to all other known examples of cdc2. Despite this strong similarity, however, Eg1 was unable to complement a yeast cdc2- mutant in Schizosaccharomyces pombe or a cdc28 mutant of Saccharomyces cerevisiae. Four Eg1 transcripts, two major and two minor, were found in Xenopus oocytes and early embryos. These RNAs appeared very early (stage I) in oogenesis and their level remained constant until the midblastula transition, at which time they declined. Eg1 RNA is found in the poly(A)+ fraction of oocytes only between the time of meiotic maturation and fertilization--that is to say, in the unfertilized egg. At fertilization the RNA loses its poly(A) tail and at the same time leaves the polyribosomes.

Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

Stability of maternal mRNA in Xenopus embryos: role of transcription and translation.

The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.

Contribution of different GC-motifs to the control of simian virus 40 late promoter activity.

During the course of lytic infection the 21-bp repeat region regulates differentially the late gene expression; a mutant deleted for this region expresses late genes either to a higher level in the absence of T antigen or to a lower level in the late phase of infection as compared to wild type (23). By analysing a series of clustered point mutations generated within the GC-motifs we show that i) mutations within motifs I and II stimulate late transcription two to three-fold, suggesting that competition for transcription machinery between early-early and late promoters is mediated by these two motifs, ii) after viral replication, simultaneous mutations within motifs IV, V and VI decrease to 23% the efficiency of late transcription, indicating that these motifs are elements of the late promoter. Moreover comparison of results presented in this paper with results published by Barrera-Saldana et al. strongly suggest that late-early and late promoters are regulated in a similar manner.

The sequence motifs that are involved in SV40 enhancer function also control SV40 late promoter activity.

The simian virus 40 (SV40) enhancer element is constituted of two domains which contain sequences important for late transcription (M. Ernoult-Lange, F. Omilli, D. O'Reilly and E. May, J. Virol. 61, 167-176, 1987). By analysing a series of clustered point mutations generated throughout the enhancer region we mapped domain I from nt 232 to 272 and domain II from nt 184 to 216. These two domains which are required for late promoter activity both in the presence and in the absence of T antigen correspond closely to the domains B and A respectively, identified for enhancer function (M. Zenke, T. Grundström, H. Matthes, M. Wintzerith, C. Schatz, A. Wildeman and P. Chambon, EMBO J., 5, 387-397, 1986). Similarly to the enhancer function the late promoter elements defined by these two domains contain multiple sequence motifs. Moreover there is a striking overlap between the sequence motifs within domain A, active for early enhancer function and those within domain II involved in efficient late transcription.

SV40 late promoter: contribution of the initiation site sequences to basal late promoter activity.

We have previously shown that the +7 to -53 element of the SV40 late promoter (nt 273 to nt 332) does not have any promoter activity, but is able to stimulate the late promoter activity of the enhancer element. The +7 to -53 element contains several late transcriptional initiation sites and we have shown that its removal results in an increase in the heterogeneity of initiation sites. Furthermore we found that inversion of the +7 to -53 element does not adversely affect the efficiency of late transcription. However, when the +7 to -53 element was inverted, transcription initiated from a single site at nt 302. In fact, we noticed that there is a consensus TATA box signal 26 nt upstream of this single site in the inverted +7 to -53 element. These results may indicate that the ability of +7 to -53 element to function in both orientations is due to the fact that, in both orientations, it possesses sequences capable of fixing the initiation sites of transcription.

Characterization of the simian virus 40 late promoter: relative importance of sequences within the 72-base-pair repeats differs before and after viral DNA replication.

We examined sequences involved in the simian virus 40 (SV40) late promoter in vivo, by using quantitative S1 nuclease analysis of a series of deletion mutants within the SV40 regulatory region. These mutants were constructed so as to place the altered promoter region in its normal position relative to the SV40 late genes. The effects of the deletions on late transcriptional activity were analyzed before and after viral DNA replication, by omitting or including SV40 large T antigen. The data show that (i) in the absence of large T antigen, the deletion of the 21-base-pair (bp) repeats results in a fourfold increase in late transcription, and (ii) the sequences within the 72-bp repeats are a component of the SV40 late promoter, acting not only before, but also after viral DNA replication. We identified two domains which contain sequences important for efficient late transcription. Domain I, at the late proximal end of each 72-bp repeat, was found to function before replication and was possibly also involved after replication. The contribution of domain II, at the late distal end of each 72-bp repeat, was much more significant after replication but only of minor importance before replication.

Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen.

We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.