Vincent Legagneux

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.

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.

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.

Multiple roles of Condensins: a complex story

Summry— Condensins are pentameric complexes that were initially described as being involved in the dynamics of chromosomes during mitosis. It has been recently established that two related complexes (Condensin I and Condensin II) contribute to this process. An increasing sum of studies, using different approaches in various organisms, leads to the paradigm that Condensins are required for the correct segregation of replicated chromosomes by cooperating somehow with Topoisomerase II in sister chromatid resolution. Depending on species and/or experimental studies, these complexes also contribute to some aspects of the assembly and compaction of mitotic chromosomes. Recent studies provided evidences that Condensins and related complexes also function in non‐mitotic processes such as replication and transcription. Biochemical studies have highlighted mechanistic aspects of Condensin function and initiated a fine functional dissection of core and regulatory subunits. However, the exact contribution of each subunit remains largely elusive as well as the functional interplay between Condensin I and Condensin II.

Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis.

ABSTRACT Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

Nucleolar association of pEg7 and XCAP-E, two members of xenopus laevis condensin complex in interphase cells

Cell cycle dynamics and localization of condensins — multiprotein complexes involved in late stages of mitotic chromosome condensation — were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to β-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

Post-transcriptional controls – adding a new layer of regulation to clock gene expression

Living organisms undergo biochemical, physiological and behavioral cycles with periods ranging from seconds to years. Cycles with intermediate periods are governed by endogenous clocks that depend on oscillating gene expression. Here we illustrate the modalities and specific functions of post-transcriptional control of gene expression (exerted on pre-mRNAs and mRNAs) in biological clocks through two examples: the circadian clock and the vertebrate somite segmentation clock, an embryonic clock with a period far below a day. We conclude that both constitutive and cyclic post-transcriptional controls underpin clock function.

Introduction to chromosome dynamics in mitosis

To ensure that the genetic information, replicated in the S‐phase of the cell cycle, is correctly distributed between daughter cells at mitosis, chromatin duplication and chromosome segregation are highly regulated events. Since the early 1980s, our knowledge of the mechanisms governing these two events has greatly increased due to the use of genetic and biochemical approaches. We present here, first, an overview of the replication process, highlighting molecular aspects involved in coupling replication with chromatin dynamics in mitosis. The second part will present the current understanding of chromosome condensation and segregation during mitosis in higher eukaryotes. Finally, we will underline the links that exist between replication and mitosis.

Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation.

CUGBP1 is an RNA-binding protein controlling alternative splicing, mRNA translation and stability. In this work we used a motif scoring approach to identify putative CUGBP1 binding sites for genes located on the human chromosome 12. This allowed us to identify the gene CD9 as a presumptive target for CUGBP1-mediated regulation. In a number of cancers, the tetraspanin CD9 is down-regulated, an event correlated with a bad prognostic. Using a combination of biochemical approaches and CUGBP1 knockdown, we showed that CUGBP1 directly controls CD9 expression.

Distribution of XCAP-E and XCAP-D2 in the Xenopus oocyte nucleus

Several antibodies were used to examine the distribution of two condensin members, XCAP-E and XCAP-D2, in the nucleus of Xenopus oocytes. XCAP-D2 was found to be associated with the lampbrush chromosomes. The chromosomal regions containing XCAP-D2 correspond precisely to domains of highly compacted chromatin, suggesting a direct contribution of XCAP-D2 in meiotic chromatin organization. In contrast, XCAP-E was found to be absent from chromosomes but was detected at a high concentration in the granular component of nucleoli. The subnucleolar localization of XCAP-E was further confirmed by double labeling using several nucleolar protein markers. The fate of nucleolar XCAP-E was also followed when changes in the nucleoli morphology were artificially induced. The apparent exclusion of XCAP-E from the ribosomal DNA and its tight association with the granular component in all preparations suggest that it might be sequestrated in nucleoli during early stages of meiosis. Interestingly, both XCAP-D2 and XCAP-E were also detected in Cajal bodies, which are organelles suspected to play a role in the assembly/modification of the RNA transcription and processing machinery. The presence of two condensins in CBs might extend such a role of assembly to chromatin macromolecular components as well.