Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins

1994 ◽  
Vol 14 (12) ◽  
pp. 7867-7875
Author(s):  
R Simon ◽  
J D Richter
Keyword(s):  
Oocyte Maturation ◽  
Untranslated Region ◽  
Early Embryo ◽  
Early Embryogenesis ◽  
Cross Linking ◽  
Wild Type ◽  
Similar Sequence ◽  
Cytoplasmic Polyadenylation ◽  
Egg Extracts ◽  
Maternal Mrnas

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.

scholarly journals Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

1994 ◽  
Vol 14 (12) ◽  
pp. 7867-7875 ◽  
Author(s):  
R Simon ◽  
J D Richter
Keyword(s):  
Oocyte Maturation ◽  
Untranslated Region ◽  
Early Embryo ◽  
Early Embryogenesis ◽  
Cross Linking ◽  
Wild Type ◽  
Similar Sequence ◽  
Cytoplasmic Polyadenylation ◽  
Egg Extracts ◽  
Maternal Mrnas

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.

scholarly journals Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes.

1990 ◽  
Vol 10 (11) ◽  
pp. 5634-5645 ◽  
Author(s):  
J Paris ◽  
J D Richter
Keyword(s):  
Complex Formation ◽  
Oocyte Maturation ◽  
Translational Control ◽  
Untranslated Regions ◽  
Stable Complex ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
Egg Extracts ◽  
Gel Mobility Shift ◽  
Rna Polyadenylation

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.

Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes

1990 ◽  
Vol 10 (11) ◽  
pp. 5634-5645
Author(s):  
J Paris ◽  
J D Richter
Keyword(s):  
Complex Formation ◽  
Oocyte Maturation ◽  
Translational Control ◽  
Untranslated Regions ◽  
Stable Complex ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
Egg Extracts ◽  
Gel Mobility Shift ◽  
Rna Polyadenylation

Early embryonic development in Xenopus laevis is programmed in part by maternally derived mRNAs, many of which are translated at the completion of meiosis (oocyte maturation). Polysomal recruitment of at least one of these mRNAs, G10, is regulated by cytoplasmic poly(A) elongation which, in turn, is dependent upon the cytoplasmic polyadenylation element (CPE) UUUUUUAUAAAG and the hexanucleotide AAUAAA (L. L. McGrew, E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter, Genes Dev. 3:803-815, 1989). We have investigated whether sequences similar to the G10 RNA CPE that are present in other RNAs could also be responsible for maturation-specific polyadenylation. B4 RNA, which encodes a histone H1-like protein, requires a CPE of the sequence UUUUUAAU as well as the polyadenylation hexanucleotide. The 3' untranslated regions of Xenopus c-mos RNA and mouse HPRT RNA also contain U-rich CPEs since they confer maturation-specific polyadenylation when fused to Xenopus B-globin RNA. Polyadenylation of B4 RNA, which occurs very early during maturation, is limited to 150 residues, and it is this number that is required for polysomal recruitment. To investigate the possible diversity of factors and/or affinities that might control polyadenylation, egg extracts that faithfully adenylate exogenously added RNA were used in competition experiments. At least one factor is shared by B4 and G10 RNAs, although it has a much greater affinity for B4 RNA. Additional experiments demonstrate that an intact CPE and hexanucleotide are both required to compete for the polyadenylation apparatus. Gel mobility shift assays show that two polyadenylation complexes are formed on B4 RNA. Optimal complex formation requires an intact CPE and hexanucleotide but not ongoing adenylation. These data, plus additional RNA competition studies, suggest that stable complex formation is enhanced by an interaction of the trans-acting factors that bind the CPE and polyadenylation hexanucleotide.

Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 579-588 ◽  
Author(s):  
M.E. Lieberfarb ◽  
T. Chu ◽  
C. Wreden ◽  
W. Theurkauf ◽  
J.P. Gergen ◽  
...  
Keyword(s):  
Mrna Translation ◽  
Vital Role ◽  
Wild Type ◽  
Regulatory Pathway ◽  
Cytoplasmic Polyadenylation ◽  
Maternal Mrna ◽  
Independent Mechanism ◽  
Maternal Mrnas ◽  
Early Metazoan

Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, we have examined a collection of Drosophila female-sterile mutants for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of bicoid and Toll mRNAs. Cortex embryos contain a bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly(A)) tail. Injection of polyadenylated bicoid mRNA into cortex embryos allows translation demonstrating that insufficient polyadenylation prevents endogenous bicoid mRNA translation. In contrast nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. Cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development.

scholarly journals Evidence for involvement of 3'-untranslated region in determining angiotensin II receptor coupling specificity to G-protein

2003 ◽  
Vol 370 (2) ◽  
pp. 631-639 ◽  
Author(s):  
Thomas J. THEKKUMKARA ◽  
Stuart L. LINAS
Keyword(s):  
G Protein ◽  
Untranslated Region ◽  
Chinese Hamster ◽  
Cell Number ◽  
Cross Linking ◽  
Leucine Incorporation ◽  
Wild Type ◽  
Receptor Coupling ◽  
At1a Receptor ◽  
Mrna Binding

The mRNA 3′-untranslated region (3′-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT1A) with and without 3′-UTR differed in AT1A mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT1A 3′-UTR probe identified a major mRNA-binding protein complex of 55kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT1A receptor 3′-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT1A 3′-UTR sequence ‘decoy’ to compete with the native receptor 3′-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT1A receptor and [125I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT1A receptors in WB (Bmax = 2.7pmol/mg of protein, Kd = 0.56nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3′-UTR decoy-expressing cells, confirming the role of 3′-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [3H]thymidine and [3H]leucine incorporation respectively. Activation of [3H]thymidine and [3H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT1 receptor coupling with G-protein αi. We also delineated that functional coupling of AT1A receptor with G-protein αi is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein αi activation. In contrast with wild-type cells, stable expression of the 3′-UTR ‘decoy’ produced AngII-stimulated protein synthesis and cellular hypertrophy as demonstrated by a significant increase in [3H]leucine incorporation and no increase in [3H]thymidine incorporation and cell number. Furthermore, [125I]AngII cross-linking and immunoprecipitation studies using specific G-protein α antibodies showed that in wild-type cells, the AT1A receptor coupled with G-protein αi, whereas in cells expressing the 3′-UTR ‘decoy’, the AT1A receptor coupled with G-protein αq. These findings indicate that the 3′-UTR-mediated changes in receptor function may be mediated in part by a switch from G-protein αi to G-protein αq coupling of the receptor. Our results suggest that the 3′-UTR-mediated post-transcriptional modification of the AT1A receptor is critical for regulating tissue-specific receptor functions.

scholarly journals Cloning and characterization of a Xenopus poly(A) polymerase.

1995 ◽  
Vol 15 (3) ◽  
pp. 1422-1430 ◽  
Author(s):  
F Gebauer ◽  
J D Richter
Keyword(s):  
Oocyte Maturation ◽  
Biochemical Properties ◽  
Cytoplasmic Polyadenylation ◽  
Cytoplasmic Enzyme ◽  
Amino Terminal ◽  
Early Embryos ◽  
Egg Extracts ◽  
Localization Signal ◽  
The One

During oocyte maturation and early embryogenesis in Xenopus laevis, the translation of several mRNAs is regulated by cytoplasmic poly(A) elongation, a reaction catalyzed by poly(A) polymerase (PAP). We have cloned, sequenced, and examined several biochemical properties of a Xenopus PAP. This protein is 87% identical to the amino-terminal portion of bovine PAP, which catalyzes the nuclear polyadenylation reaction, but lacks a large region of the corresponding carboxy terminus, which contains the nuclear localization signal. When injected into oocytes, the Xenopus PAP remains concentrated in the cytoplasm, suggesting that it is a specifically cytoplasmic enzyme. Oocytes contain several PAP mRNA-related transcripts, and the levels of at least the one encoding the putative cytoplasmic enzyme are relatively constant in oocytes and early embryos but decline after blastulation. When expressed in bacteria and purified by affinity and MonoQ-Sepharose chromatography, the protein has enzymatic activity and adds poly(A) to a model substrate. Importantly, affinity-purified antibodies directed against Xenopus PAP inhibit cytoplasmic polyadenylation in egg extracts. These data suggest that the PAP described here could participate in cytoplasmic polyadenylation during Xenopus oocyte maturation.

scholarly journals Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation

2012 ◽  
Vol 445 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Kyle Friend ◽  
Matthew Brook ◽  
F. Betül Bezirci ◽  
Michael D. Sheets ◽  
Nicola K. Gray ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Binding Protein ◽  
Xenopus Laevis Oocytes ◽  
Protein Activity ◽  
Cytoplasmic Polyadenylation ◽  
Post Translational Modifications ◽  
Maternal Mrnas ◽  
Insight Into

Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo.

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

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1193-1202
Author(s):  
V. Legagneux ◽  
P. Bouvet ◽  
F. Omilli ◽  
S. Chevalier ◽  
H.B. Osborne
Keyword(s):  
Untranslated Region ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Untranslated Regions ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein ◽  
Maternal Mrnas ◽  
Post Transcriptional Regulation

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.

Sequential waves of polyadenylation and deadenylation define a translation circuit that drives meiotic progression

2008 ◽  
Vol 36 (4) ◽  
pp. 665-670 ◽  
Author(s):  
Eulàlia Belloc ◽  
Maria Piqué ◽  
Raúl Méndez
Keyword(s):  
Untranslated Region ◽  
Translational Regulation ◽  
Feedback Loops ◽  
Quiescent State ◽  
Cytoplasmic Polyadenylation ◽  
Meiotic Progression ◽  
Negative Feedback Loops ◽  
Positive And Negative Feedback ◽  
Maternal Mrnas ◽  
Combinatorial Code

The maternal mRNAs that drive meiotic progression in oocytes contain short poly(A) tails and it is only when these tails are elongated that translation takes place. Cytoplasmic polyadenylation requires two elements in the 3′-UTR (3′-untranslated region), the hexanucleotide AAUAAA and the CPE (cytoplasmic polyadenylation element), which also participates in the transport and localization, in a quiescent state, of its targets. However, not all CPE-containing mRNAs are activated at the same time during the cell cycle, and polyadenylation is temporally and spatially regulated during meiosis. We have recently deciphered a combinatorial code that can be used to qualitatively and quantitatively predict the translational behaviour of CPE-containing mRNAs. This code defines positive and negative feedback loops that generate waves of polyadenylation and deadenylation, creating a circuit of mRNA-specific translational regulation that drives meiotic progression.

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