H. Beverley Osborne

AU-rich elements and associated factors: are there unifying principles?

The control of mRNA stability is an important process that allows cells to not only limit, but also rapidly adjust, the expression of regulatory factors whose over expression may be detrimental to the host organism. Sequence elements rich in A and U nucleotides or AU-rich elements (AREs) have been known for many years to target mRNAs for rapid degradation. In this survey, after briefly summarizing the data on the sequence characteristics of AREs, we present an analysis of the known ARE-binding proteins (ARE-BP) with respect to their mRNA targets and the consequences of their binding to the mRNA. In this analysis, both the changes in mRNA stability and the lesser studied effects on translation are considered. This analysis highlights the multitude of mRNAs bound by one ARE-BP and conversely the large number of ARE-BP that associate with any particular ARE-containing mRNA. This situation is discussed with respect to functional redundancies or antagonisms. The potential relationship between mRNA stability and translation is also discussed. Finally, we present several hypotheses that could unify the published data and suggest avenues for future research.

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.

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.

Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis

The distribution of four specific RNA species between the poly(A)+ and poly(A)- fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c-raf mRNA and the last one to RNA revealed by a mouse ornithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A)+ population. During the same period, the other two RNAs are deadenylated and these new poly(A)- RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.

Tethering of proteins to RNAs by bacteriophage proteins

Many steps in the control of gene expression are dependent on RNA‐binding proteins, most of which are bi‐functional, in as much as they both bind to RNA and interact with other protein partners in a functional complex. A powerful approach to study the functional properties of these proteins in vivo, independently of their RNA‐binding ability, is to attach or tether them to specifically engineered reporter mRNAs whose fate can be easily followed. Two tethering systems have been mainly used in eukaryotic cells, namely the MS2 coat protein system and the lambda N—B box system. In this review, we firstly describe several studies in which these tethering systems have been used and provide an overview of these applications. We next describe the major features of these two systems, and, finally, we highlight a number of points that should be considered when designing experiments using this approach.

East of EDEN was a poly(A) tail.

Post‐transcriptional regulations of gene expression (control of mRNA stability and translation) play a central role in achieving cellular functions. In a large number of cases, post‐transcriptional regulations are dependent on mRNA poly(A) tails, as mRNAs with a long poly(A) tail are generally much more stable and actively translated than deadenylated mRNAs. In this review, we will discuss the activities that modify poly(A) tail lengths in Xenopus oocytes and embryos. We will particularly focus on one activity, the “EDEN” mechanism, that provokes specific poly(A) tail shortening rapidly after fertilization. EDEN‐dependent deadenylation is mediated by the specific binding of a protein, EDEN‐BP. The EDEN mechanism will be compared with several other mechanisms that provoke deadenylation in a large variety of species. The proposal that the EDEN mechanism is probably a mechanism of widespread importance in the metazoan world will be discussed.

Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis

The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3′ untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3′ UTR for an EDEN-BP-dependent deadenylation after fertilization.

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.

Polyamine levels during Xenopus laevis oogenesis: a role in oocyte competence to meiotic resumption

The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.

Our favourite alternative splice site.

Alternative splicing is a widespread mechanism in mammals that generates several mRNAs from one gene, thereby creating genetic diversity of the genome. Variant splice patterns are often specific to different stages of development or particular tissues, and alternative splicing defects are being more frequently detected in genetic diseases and cancers. The increasingly important role of alternative splicing in the function and the regulation of cellular process makes it critical to have an easy‐to‐use data repository for the biological and medical research communities. We have compared web resources that give access to information on alternatively spliced genes, and the FAST DB (Friendly Alternative Splicing and Transcripts DataBase) site came out as our favourite.