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.     

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.     

The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element

We reported in our previous work that the EDEN-dependent translational repression of maternal mRNAs was conserved between Drosophila and Xenopus. In Xenopus, this repression is achieved through the binding of EDEN to the Bruno-like factor, EDEN-BP. We show in the present work that the Drosophila Bruno paralogue, the 45 kDa Bru-3 protein (p45), binds specifically to the EDEN element and acts as a homodimer. We describe for the first time a previously undetected 67 amino acid domain, found in the divergent linker region, the lsm domain (lsm stands for linker-specific motif). We propose that the presence of this domain in a subset of the Bruno-like proteins, including Bru-3, EDEN-BP and CUG-BP but not Bruno nor its other paralogue Bru-2, might be responsible for specific RNA recognition. Interestingly, comparative structural analyses using threaders and molecular modelling suggest that the new domain might be distantly related to the first RNA recognition motif of the Drosophila sex-lethal protein (sxl). The phylogenetic analyses and the experimental data based on its specific binding to the EDEN element support the conclusion that Bru-3 is an EDEN-BP/CUG-BP orthologue.     

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.     

An analysis of the sequence requirements of EDEN-BP for specific RNA binding

EDEN-BP (embryo deadenylation element-binding protein) binds specifically to the EDEN motif in the 3′-untranslated regions of maternal mRNAs and targets these mRNAs for deadenylation and translational repression in Xenopus laevis embryos. EDEN-BP contains three RNA recognition motifs (RRMs) and is related to the elav family of RNA-binding proteins. In the present study we show that the two N-terminal RRMs of EDEN-BP are necessary for the interaction with EDEN as well as a part of the linker region (between RRM2 and RRM3). Using a band shift assay we show that two different complexes are formed according to the size and, therefore, the functional nature of the EDEN motif. Finally, we show that EDEN-BP can form a dimer in a two-hybrid assay. Accordingly, we suggest that the functional configuration of EDEN-BP is a dimer.     

Translational control of maskin mRNA by its 3' untranslated region

BACKGROUND INFORMATION: Maskin is a member of the TACC (transforming acidic coiled-coil) domain proteins found in Xenopus laevis oocytes and embryos. It has been implicated in the co-ordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. RESULTS: In the present study, we report that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3'-UTR (untranslated region) of maskin can confer the translational regulation to a reporter mRNA, and so can the 3'-UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV cross-linking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3'-UTRs. As reported previously, CPEB [CPE (cytoplasmic polyadenylation element)-binding protein] binds to the cyclin B1 3'-UTR, but its binding to the maskin 3'-UTR is minimal. By RNA affinity chromatography and MS, we identified the EDEN-BP [EDEN (embryonic deadenylation element)-binding protein] as one of the proteins binding to both the maskin and the cyclin B1 3'-UTRs. CONCLUSIONS: Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3'-UTRs, indicating it may be involved in the deadenylation of these mRNAs.     

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.     

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.     

PAB1 self-association precludes its binding to poly(A), thereby accelerating CCR4 deadenylation in vivo

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.     

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.     

CUG-BP binds to RNA substrates and recruits PARN deadenylase

CUG-BP is the human homolog of the Xenopus EDEN-BP, which was shown previously to bind to mRNAs, such as c-mos, that exhibit rapid deadenylation following fertilization of the oocyte. While several studies have focused on roles of CUG-BP as a splicing or translation regulator in mammalian cells, its role in mRNA decay has not been examined in detail. Here, we have used an in vitro deadenylation assay to dissect the function of CUG-BP in the decay of two ARE-containing mRNAs: c-fos and TNFalpha. CUG-BP binds specifically to both of these RNAs and stimulates poly(A) shortening by PARN. Moreover, CUG-BP interacts with PARN in extracts by coimmunoprecipitation, and this interaction can be recapitulated using recombinant proteins. CUG-BP, therefore, is the first RNA-binding protein shown to directly recruit a deadenylase to an RNA substrate.     

Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability.     

microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.     

The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation

The poly(A)-binding protein PAB1 from the yeast Saccharomyces cerevisiae plays an important role in controlling mRNA deadenylation rates. Deletion of either its RRM1 or proline-rich domain (P domain) severely restricts deadenylation and slows mRNA degradation. Because these large deletions could be having unknown effects on the structure of PAB1, different strategies were used to determine the importance of the RRM1 and P domains to deadenylation. Since the P domain is quite variable in size and sequence among eukaryotes, P domains from two human PABPCs and from Xenopus were substituted for that of PAB1. The resultant PAB1 hybrid proteins, however, displayed limited or no difference in mRNA deadenylation as compared with PAB1. In contrast to the P domain, the RRM1 domain is highly conserved across species, and a systematic mutagenesis of the RRM1 domain was undertaken to identify its functional regions. Several mutations along the RNA-binding surface of RRM1 inhibited deadenylation, whereas one set of mutations on its exterior non-RNA binding surface shifted deadenylation from a slow distributive process to a rapid processive deadenylation. These results suggest that the RRM1 domain is the more critical region of PAB1 for controlling deadenylation and consists of at least two distinguishable functional regions.     

HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA

The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.