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.

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.

scholarly journals Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

1992 ◽  
Vol 12 (1) ◽  
pp. 413-421 ◽  
Author(s):  
P Cortes ◽  
O Flores ◽  
D Reinberg
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Stable Complex ◽  
Mobility Shift ◽  
Yeast Saccharomyces Cerevisiae ◽  
Gel Mobility Shift Assay ◽  
Two Factors ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Tfiid Complex

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

Hybridization properties of oligoarabinonucleotides

1994 ◽  
Vol 72 (3) ◽  
pp. 909-918 ◽  
Author(s):  
Paul Apostolos Giannaris ◽  
Masad José Damha
Keyword(s):  
Solid Phase ◽  
Stable Complex ◽  
Mobility Shift ◽  
Complementary Dna ◽  
Melting Temperatures ◽  
Snake Venom Phosphodiesterase ◽  
Dna And Rna ◽  
Negative Effect ◽  
Gel Mobility Shift ◽  
Rna Complexes

3′-Phosphoramidite derivatives of arabinocytidine, arabinoadenosine, and arabinouridine were prepared and used for the solid-phase synthesis of oligoarabinonucleotides (arabinonucleic acid, or ANA). Thermal denaturation analysis and gel mobility shift assays were used to investigate the interaction between ANA and complementary DNA and RNA. In general, the ANA/DNA and ANA/RNA complexes exhibited melting temperatures comparable to those of the corresponding DNA/DNA and DNA/RNA complexes. Thus the inversion of stereochemistry at the C2′ of ribonucleotides does not have a negative effect on interaction with natural sequences. In fact, in complexes with poly dT, oligoarabinoadenylates displayed greater hybridization affinity than oligoriboadenylates. In summary, we observed that (i) ara(Ap)7A interacted favourably with poly rU and poly dT, (ii) ara(Cp)7C formed a stable complex with poly rG; (iii) ara(Up)7U did not bind with complementary poly rA; and (iv) the mixed oligoarabinonucleotide ara(UCU UCC CUC UCC C) formed complexes with complementary DNA and RNA. Hybridization was observed between the phosphorothioatelinked arabinoadenylate ara(Aps)7A and poly rU and poly dT; however, this binding was weaker than that between the phosphorothioate-linked deoxyriboadenylate d(Aps)7A and poly rU and poly dT. Both ara(Aps)7A and its deoxy analog d(Aps)7A displayed significant and similar resistance to digestion by snake venom phosphodiesterase.

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 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.

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ

1992 ◽  
Vol 12 (1) ◽  
pp. 413-421
Author(s):  
P Cortes ◽  
O Flores ◽  
D Reinberg
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Stable Complex ◽  
Mobility Shift ◽  
Yeast Saccharomyces Cerevisiae ◽  
Gel Mobility Shift Assay ◽  
Two Factors ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Tfiid Complex

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

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.

Timely translation during the mouse oocyte-to-embryo transition

Development ◽  
2000 ◽  
Vol 127 (17) ◽  
pp. 3795-3803 ◽  
Author(s):  
B. Oh ◽  
S. Hwang ◽  
J. McLaughlin ◽  
D. Solter ◽  
B.B. Knowles
Keyword(s):  
Oocyte Maturation ◽  
Translational Control ◽  
Expressed Sequence Tag ◽  
Control Program ◽  
Mobility Shift ◽  
Cell Stage ◽  
Reading Frame ◽  
Transcript Stability ◽  
The Stability ◽  
Beta Galactosidase

In the mouse, completion of oocyte maturation and the initiation of preimplantation development occur during transcriptional silence and depend on the presence and translation of stored mRNAs transcribed in the growing oocyte. The Spin gene has three transcripts, each with an identical open reading frame and a different 3′ untranslated region (UTR). (Beta)-galactosidase-tagged reporter transcripts containing each of the different Spin 3′UTRs were injected into oocytes and zygotes and (beta)-galactosidase activity was monitored. Results from these experiments suggest that differential polyadenylation and translation occurs at two critical points in the oocyte-to-embryo transition - upon oocyte maturation and fertilization - and is dependent on sequences in the 3′UTR. The stability and mobility shifts of ten other maternal transcripts were monitored by reprobing a northern blot of oocytes and embryos collected at 12 hour intervals after fertilization. Some are more stable than others and the upward mobility shift associated with polyadenylation correlates with the presence of cytoplasmic polyadenylation elements (CPEs) within about 120 nucleotides of the nuclear polyadenylation signal. A survey of the 3′ UTRs of expressed sequence tag clusters from a mouse 2-cell stage cDNA library indicates that about one third contain CPEs. We suggest that differential transcript stability and a translational control program can supply the diversity of protein products necessary for oocyte maturation and the initiation of development.

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