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.     

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.     

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.     

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.     

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.     

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.     

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.     

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.     

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.     

Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR

AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.     

Cap-dependent deadenylation of mRNA

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.     

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.     

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.