Luc Paillard

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.

CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

The exon junction complex (EJC) is a central effector of the fate of mRNAs, linking nuclear processing to mRNA transport, translation and surveillance. However, little is known about its transcriptome-wide targets. We used cross-linking and immunoprecipitation methods coupled to high-throughput sequencing (CLIP-seq) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks that are located mainly in spliced mRNAs. Most expressed exons harbor peaks either in the canonical EJC region, located ~24 nucleotides upstream of exonic junctions, or in other noncanonical regions. Notably, both of these types of peaks are preferentially associated with unstructured and purine-rich sequences containing the motif GAAGA, which is a potential binding site for EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.

Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1−/− mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1−/− males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1−/− males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

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.

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

Abstract 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.

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.

Reverse genetics in eukaryotes

Reverse genetics consists in the modification of the activity of a target gene to analyse the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centred on genome alterations. Mutations produced by random chemical or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other two approaches are centred on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metabolism or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to one of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome‐wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post‐genomic era.

EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation.

EDEN-BP is a Xenopus RNA-binding protein that triggers deadenylation [poly(A) tail shortening], and thereby translational repression and degradation, of a subset of maternal mRNAs soon after fertilization. We show here that this factor is expressed in the presomitic mesoderm of older embryos, the site where somitic segmentation takes place. Inhibiting EDEN-BP function using either antisense morpholino oligonucleotides or neutralizing antibodies leads to severe defects in somitic segmentation, but not myotomal differentiation. This is associated with defects in the expression of segmentation markers belonging to the Notch signalling pathway in the presomitic mesoderm. We show by a combination of approaches that the mRNA encoding XSu(H), a protein that plays a central role in Notch signalling, is regulated by the EDEN-BP pathway. Accordingly, XSu(H) is overexpressed in EDEN-BP knock-down embryos, and overexpressing XSu(H) causes segmentation defects. We finally give data indicating that, in addition to XSu(H), other segmentation RNAs are a target for EDEN-BP. These results show that EDEN-BP-dependent post-transcriptional regulation of gene expression is required for the process of somitic segmentation.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms—in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3′ untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.