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At least 19 recordsLinked to original sources

PubMed1998

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.

Animals↗

PubMed2001

c-Jun ARE targets mRNA deadenylation by an EDEN-BP (embryo deadenylation element-binding protein)-dependent pathway.

In mammalian cells, certain mRNAs encoding cytokines or proto-oncogenes are especially unstable, because of the presence of a particular sequence element in their 3'-untranslated region named ARE (A/U-rich element). AREs cause this instability by provoking the rapid shortening of the poly(A) tail of the mRNA. The deadenylation of mRNAs mediated by AREs containing repeats of the AUUUA motif (class I/II AREs) is conserved in Xenopus embryos. Here, we first extend these observations by showing that c-Jun ARE, a representative of class III (non-AUUUA) AREs, also provokes the deadenylation of a reporter RNA in Xenopus embryos. Next, by immunodepletion and immunoneutralization experiments, we show that, in Xenopus, the rapid deadenylation of RNAs that contain the c-Jun ARE, but not an AUUUA ARE, requires EDEN-BP. This RNA-binding protein was previously shown to provoke the rapid deadenylation of certain Xenopus maternal RNAs. Finally, we show that CUG-BP, the human homologue of EDEN-BP, specifically binds to c-Jun ARE. Together, these results identify CUG-BP as a plausible deadenylation factor responsible for the post-transcriptional control of c-Jun proto-oncogene mRNA in mammalian cells.

3' Untranslated Regions↗

PubMedDate not supplied

A functional deadenylation assay identifies human CUG-BP as a deadenylation factor.

CUG-BP is a human nuclear and cytoplasmic RNA-binding protein. A role in the control of alternative splicing has been reported, but to date no cytoplasmic function for this protein has been demonstrated. A close sequence homolog of CUG-BP is EDEN-BP that is required for the specific cytoplasmic poly(A) tail shortening of certain mRNAs after fertilization of Xenopus eggs. Here, we show that human CUG-BP and Xenopus EDEN-BP have very similar RNA-binding specificities. In addition, we use a deadenylation assay to show that CUG-BP is able to act as a deadenylation factor. In contrast, a mutant form of CUG-BP, though still able to bind to RNA with a specificity similar to that of wild-type CUG-BP, does not act as a deadenylation factor. It is suggested that the CUG expansion associated with Type 1 myotonic dystrophy can affect the function or the activity of CUG-BP, leading to a trans-dominant effect on normal RNA processing. The results presented here identify CUG-BP-dependent deadenylation as a potential cytoplasmic target for this trans-dominant effect.

CELF1 Protein↗

PubMed2003

Deadenylation of interferon-beta mRNA is mediated by both the AU-rich element in the 3'-untranslated region and an instability sequence in the coding region.

Viral infection of fibroblastic and endothelial cells leads to the transient synthesis of interferon-beta (IFN-beta). The down-regulation of IFN-beta synthesis after infection results both from transcriptional repression of the IFN-beta gene and rapid degradation of mRNA. As with many cytokine mRNAs, IFN-beta mRNA contains an AU-rich element (ARE) in its 3'-untranslated region (UTR). AREs are known to mediate mRNA deadenylation and destabilization. Depending on the class of ARE, deadenylation was shown to occur through synchronous or asynchronous mechanisms. In this study, we analysed IFN-beta mRNA deadenylation in natural conditions of IFN-beta synthesis, e.g., after viral infection. We show that human IFN-beta mRNA follows an asynchronous deadenylation pathway typical of a mRNA containing a class II ARE. A deletion analysis of the IFN-beta natural transcript demonstrates that poly(A) shortening can be mediated by the ARE but also by a 32 nucleotide-sequence located in the coding region, that was identified previously as an instability determinant. In fact, these elements are able to act independently as both of them have to be removed to abrogate mRNA deadenylation. Our data also indicate that deadenylation occurs independently of mRNA translation. Moreover, we show that deadenylation of IFN-beta mRNA is not under the control of viral infection as IFN-beta mRNA derived from a constitutively expressed gene cassette is deadenylated in absence of viral infection. Finally, an unidentified nuclear event appears to be a prerequisite for IFN-beta mRNA deadenylation as IFN-beta mRNA introduced directly into the cytoplasm does not undergo deadenylation. In conclusion, our study demonstrates that IFN-beta mRNA poly(A) shortening is under the control of two cis-acting elements recruiting a deadenylating machinery whose activity is independent of translation and viral infection but might require a nuclear event.

3' Untranslated Regions↗

PubMed1998

AUUUA sequences direct mRNA deadenylation uncoupled from decay during Xenopus early development.

To study the regulation of AUUUA-mediated RNA deadenylation and destabilization during Xenopus early development, we microinjected chimeric mRNAs containing Xenopus or mammalian 3' untranslated region (3'-UTR) sequences into Xenopus oocytes, mature eggs, or fertilized embryos. We found that the AU-rich elements (ARE) of Xenopus c-myc II and the human granulocyte-macrophage colony-stimulating factor gene (GMCSF) both direct deadenylation of chimeric mRNAs in an AUUUA-dependent manner. In the case of the Xenopus c-myc II ARE, mutation of a single AUUUA within an absolutely conserved 11-nucleotide region in c-myc 3'-UTRs prevents ARE-mediated deadenylation. AUUUA-specific deadenylation appears to be developmentally regulated: low deadenylation activity is observed in the oocyte, whereas rapid deadenylation occurs following egg activation or fertilization. Deadenylation results in the accumulation of stable deadenylated RNAs that become degraded only following mid-blastula transition. We conclude that ARE-mediated mRNA deadenylation can be uncoupled from ARE-mediated mRNA decay and that AUUUAs directly signal deadenylation during Xenopus early development.

3' Untranslated Regions↗

PubMed2003

Regulation of EDEN-dependent deadenylation of Aurora A/Eg2-derived mRNA via phosphorylation and dephosphorylation in Xenopus laevis egg extracts.

Deadenylation is an intimate part of the post-transcriptional regulation of maternal mRNAs in embryos. EDEN-BP is so far the only known member of a complex regulating the deadenylation of maternal mRNA in Xenopus laevis embryos in a manner that is dependent on the 3'-untranslated region called EDEN (embryo deadenylation element). In this report, we show that calcium activation of cell-free extracts triggers EDEN binding protein (EDEN-BP) dephosphorylation and concomitant deadenylation of a chimeric RNA bearing Aurora A/Eg2 EDEN sequence. Deadenylation of mRNA deprived of EDEN sequence (default deadenylation) does not change with egg activation. Kinase and phosphatase inhibitors downregulate EDEN-dependent deadenylation but they do not substantially influence default deadenylation. Using indestructible Delta90 cyclin B to revert interphase extracts to the M-phase, we show that modulation of EDEN-dependent deadenylation is independent of M-phase promoting factor (MPF) activity. These results suggest that the increase in EDEN-dependent deadenylation following egg activation is achieved, at least partially, via dephosphorylation and/or phosphorylation of regulatory proteins, including EDEN-BP dephosphorylation. This regulation proceeds in a manner independent from MPF inactivation.

3' Untranslated Regions↗

PubMed1995

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.

Animals↗

PubMed2006

Rapid ATP-dependent deadenylation of nanos mRNA in a cell-free system from Drosophila embryos.

Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has recently also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, we have developed a cell-free system from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3'-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4.NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation.

3' Untranslated Regions↗

PubMed1992

Maturation-specific deadenylation in Xenopus oocytes requires nuclear and cytoplasmic factors.

During the meiotic maturation of Xenopus oocytes, maternal mRNAs that lack a cytoplasmic polyadenylation element are deadenylated and translationally inactivated. In this report, we have characterized the regulation of poly(A) removal during maturation. Deadenylation in vivo is detected only after germinal vesicle breakdown and does not require de novo protein synthesis. Enucleated oocytes do not deadenylate either endogenous or microinjected RNAs upon maturation, indicating that a nuclear component is required for poly(A) removal. Whole cell extracts prepared from both immature and mature oocytes deadenylate exogenous RNA substrates in vitro. Deadenylation activity is not detected in isolated nuclear or cytoplasmic extracts obtained from immature oocytes, but is reconstituted when these fractions are combined in vitro. These results indicate that the factors required for deadenylation activity are present in immature oocytes, but that poly(A) removal is prevented by the sequestration of one or more of these components within the nucleus. Maturation-specific deadenylation of maternal mRNAs occurs upon the release of nuclear factors into the cytoplasm at germinal vesicle breakdown.

Adenosine↗

PubMed2006

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.

Adenosine Monophosphate↗

PubMed2003

p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation.

AU-rich elements (AREs) in 3'-untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated beta-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the beta-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the beta-globin reporter mRNA either lacking an ARE or containing the c-Myc 3'-untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.

Base Sequence↗

PubMed1990

Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control.

The meiotic maturation of Xenopus oocytes initiates significant changes in the translation of a number of maternal mRNAs that coincide with alterations in their polyadenylation states. A considerable number of maternal mRNAs are deadenylated in mature oocytes, thereby reducing their translational efficiencies. In this report we demonstrate that deadenylation does not require specific cis-acting sequences. Polyadenylated RNAs derived from either ribosomal protein or beta-globin mRNAs, or that contain non-mRNA-derived sequences, are deadenylated in mature oocytes. Translation of a substrate RNA is not required for its deadenylation. G10 mRNA is representative of a class of mRNAs that is translationally activated at maturation and contains the cytoplasmic polyadenylation element (U)6AU. A deletion mutant G10 transcript that lacks the (U)6AU element is not polyadenylated in mature oocytes but is deadenylated instead. Insertion of the (U)6AU element into the 3'-untranslated region of the ribosomal protein L1 mRNA is sufficient to prevent both its deadenylation and polysomal release in mature oocytes. These results indicate that the deadenylation and translational inactivation of maternal mRNAs during Xenopus oocyte maturation occur by a default pathway in which transcripts lacking a cytoplasmic polyadenylation element undergo poly(A) removal.

Animals↗

PubMed2001

A novel embryonic poly(A) binding protein, ePAB, regulates mRNA deadenylation in Xenopus egg extracts.

An in vitro system that recapitulates the in vivo effect of AU-rich elements (AREs) on mRNA deadenylation has been developed from Xenopus activated egg extracts. ARE-mediated deadenylation is uncoupled from mRNA body decay, and the rate of deadenylation increases with the number of tandem AUUUAs. A novel ARE-binding protein called ePAB (for embryonic poly(A)-binding protein) has been purified from this extract by ARE affinity selection. ePAB exhibits 72% identity to mammalian and Xenopus PABP1 and is the predominant poly(A)-binding protein expressed in the stage VI oocyte and during Xenopus early development. Immunodepletion of ePAB increases the rate of both ARE-mediated and default deadenylation in vitro. In contrast, addition of even a small excess of ePAB inhibits deadenylation, demonstrating that the ePAB concentration is critical for determining the rate of ARE-mediated deadenylation. These data argue that ePAB is the poly(A)-binding protein responsible for stabilization of poly(A) tails and is thus a potential regulator of mRNA deadenylation and translation during early development.

Adenosine Monophosphate↗

PubMed1997

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.

Adenosine Monophosphate↗

PubMed2005

The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p.

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

3' Untranslated Regions↗

PubMed2006

PUF protein-mediated deadenylation is catalyzed by Ccr4p.

PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Binding Sites↗

PubMed1998

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.

Adenine↗

PubMed1993

A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation.

To determine pathways of mRNA turnover in yeast, we have followed the poly(A) tail removal and degradation of a pulse of newly synthesized transcripts from four different genes. Before decay of both stable and unstable mRNAs initiated, there was a temporal lag during which the poly(A) tail was deadenylated to an oligo(A) length. Altering the deadenylation rate of an mRNA led to a corresponding change in the length of this lag. The rate of deadenylation and the stability of the oligo(A) species varied between mRNAs, explaining the differences in mRNA half-lives. To examine how the transcript body was degraded following deadenylation, we used the strategy of inserting strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the 3' UTR of mRNAs. Fragments lacking the 5' portion of two different mRNAs accumulated after deadenylation as full-length mRNA levels decreased. Therefore, these results define an mRNA decay pathway in which deadenylation leads to either internal cleavage or decapping followed by 5'-->3' exonucleolytic degradation of the mRNA.

Base Sequence↗