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Biomedical subjects

H Beverley Osborne

Publications and source records attributed to H Beverley Osborne.

At least 19 recordsLinked to original sources
PubMed2006

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.

3' Untranslated Regions↗
PubMed2006

Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice.

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.

Animals↗
PubMed2006

Liposome-mediated RNA transfection should be used with caution.

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

Fluorescent Dyes↗
PubMed2006

Protein expression is increased by a class III AU-rich element and tethered CUG-BP1.

In mammalian somatic cells, the post-transcriptional control of cytokine or proto-oncogene expression is often achieved by factors binding to sequence elements in the 3' untranslated region (3'UTR). The most studied are the AU-rich elements (ARE) that have been divided into three classes. Here, we show that in mammalian cells, the presence of the class III c-jun ARE in the 3'UTR of a reporter mRNA enhanced reporter protein expression. In contrast, the presence of a class II ARE in the 3'UTR decreased reporter protein expression. CUG-BP1/CELF1 is able to bind c-jun ARE. Protein expression was enhanced similarly to what was observed for c-jun ARE when the reporter mRNA contained a synthetic CUG-BP1/CELF1-binding site, or when this protein was tethered to the 3'UTR of a reporter mRNA. These results reveal an unexpected complexity of ARE-mediated post-transcriptional regulations, and indicate a function for CUG-BP1/CELF1 in class III ARE directed regulations.

3' Untranslated Regions↗
PubMed2006

Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray.

Cytoplasmic control of the adenylation state of mRNAs is a critical post-transcriptional process involved in the regulation of mRNAs stability and translational efficiency. The early development of Xenopus laevis has been a major model for the study of such regulations. We describe here a microarray analysis to identify mRNAs that are regulated by changes in their adenylation state during oogenesis and early development of the diploid frog Xenopus tropicalis. The microarray data were validated using qRT-PCR and direct analysis of the adenylation state of endogenous maternal mRNAs during the period studied. We identified more than 500 mRNAs regulated at the post-transcriptional level among the 3000 mRNAs potentially detected by the microarray. The mRNAs were classified into nine different adenylation behavior categories. The various adenylation profiles observed during oocyte maturation and early development and the analyses of 3'-untranslated region sequences suggest that previously uncharacterized sequence elements control the adenylation behavior of the newly identified mRNAs. These data should prove useful in identifying mRNAs with important functions during oocyte maturation and early development.

3' Untranslated Regions↗
PubMed2006

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.

3' Untranslated Regions↗
PubMed2006

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.

Alternative Splicing↗
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↗
PubMed2005

Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions.

In mammals, the CELF/Bruno-like family of RNA-binding proteins contains six members. The founder members of the family are the CUG-BP1 (CELF1) and ETR-3 (CELF2) proteins. Four other members have been identified mainly by sequence similarity. The founder members were cloned or identified in a number of laboratories which has lead to a profusion of names and two separate naming systems. In addition, different members of the CELF/Bruno-like protein family have been shown to be implicated in two major post-transcriptional regulatory processes, namely the alternative splicing and the control of translation and stability of target mRNAs. Several studies have indicated a certain functional redundancy between the CELF proteins in fulfilling these functions. The multiplicity of gene names and the eventual functional redundancy is a source of potential confusion in published work. We present here a synthetic picture of the present situation and, where possible, models are proposed that can account for the data obtained in the various laboratories with different biological models. Furthermore, we have highlighted some important questions that still need to be resolved.

Alternative Splicing↗
PubMed2004

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.

Animals↗
PubMed2004

Identification of a novel Xenopus laevis poly (A) binding protein.

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.

Amino Acid Sequence↗
PubMed2003

Expression and functional dynamics of the XCAP-D2 condensin subunit in Xenopus laevis oocytes.

The 13 S condensin complex plays a crucial role in the condensation and segregation of the two sets of chromosomes during mitosis in vivo as well as in cell-free extracts. This complex, conserved from yeast to human, contains a heterodimer of structural maintenance of chromosome (SMC) family proteins and three additional non-SMC subunits. We have investigated the expression of the non-SMC condensin component XCAP-D2 in Xenopus laevis oocytes. When studied during meiotic maturation, XCAP-D2 starts to accumulate at the time of germinal vesicle breakdown and reaches its maximal amount in metaphase II oocytes. This accumulation is specifically blocked by injection of antisense oligonucleotides. XCAP-D2 antisense-injected oocytes progress normally through meiosis until metaphase II. At this stage, however, chromosomes exhibit architecture defaults, and resolution of sister chromatids is impaired. Surprisingly, in mitotic extracts made from XCAP-D2 knocked-down oocytes, sperm chromatin normally condenses into compacted chromosomes, whereas the amounts of both free and chromosome-bound XCAP-D2 are markedly reduced. This apparent discrepancy is discussed in light of current knowledge on chromosome dynamics.

5' Untranslated Regions↗
PubMed2002

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.

3' Untranslated Regions↗
PubMed2002

Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking.

BACKGROUND: Specific cis-elements and the associated trans-acting factors have been implicated in the post-transcriptional regulation of gene expression. In the era of genome wide analyses identifying novel trans-acting factors and cis-regulatory elements is a step towards understanding coordinated gene expression. UV-crosslink analysis is a standard method used to identify RNA-binding proteins. Uridine is traditionally used to radiolabel substrate RNAs, however, proteins binding to cis-elements particularly uridine poor will be weakly or not detected. We evaluate here the possibility of using UV-crosslinking with RNA substrates radiolabeled with each of the four ribonucleotides as an approach for screening for novel sequence specific RNA-binding proteins. RESULTS: The radiolabeled RNA substrates were derived from the 3'UTRs of the cloned Eg and c-mos Xenopus laevis maternal mRNAs. Specific, but not identical, uv-crosslinking signals were obtained, some of which corresponded to already identified proteins. A signal for a novel 90 kDa protein was observed with the c-mos 3'UTR radiolabeled with both CTP and GTP but not with UTP. The binding site of the 90 kDa RNA-binding protein was localised to a 59-nucleotide portion of the c-mos 3'UTR. CONCLUSION: That the 90 kDa signal was detected with RNAs radiolabeled with CTP or GTP but not UTP illustrates the advantage of radiolabeling all four nucleotides in a UV-crosslink based screen. This method can be used for both long and short RNAs and does not require knowledge of the cis-acting sequence. It should be amenable to high throughput screening for RNA binding proteins.

Journal Article↗
PubMed2001

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.

3' Untranslated Regions↗
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↗