Francis Omilli

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.

Embryo Deadenylation Element-Dependent Deadenylation Is Enhanced by a cis Element Containing AUU Repeats

ABSTRACT The deadenylation of maternal mRNAs in the Xenopusembryo is a sequence-specific process. One cis element that targets maternal mRNAs for deadenylation after fertilization is the embryo deadenylation element (EDEN). This element, composed of U/R repeats, is specifically bound by a protein, EDEN-BP. In the present study we show that the rate at which an RNA containing an EDEN is deadenylated can be increased by the presence of an additionalcis element composed of three AUU repeats. This effect was observed for a natural EDEN (c-mos) and two synthetic EDENs. Hence, the enhancement of EDEN-dependent deadenylation conferred by the (AUU)3 motif is not due to an interaction with a particular EDEN sequence. Mutation of the (AUU)3 motif abrogated the enhancement of EDEN-dependent deadenylation. These data indicate that the rate at which a specific maternal mRNA is deadenylated in Xenopus embryos is probably defined by a cross talk between multiple cis elements.

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA.

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, we searched for additional PBX1 partners that might be involved in this process. Using a two hybrid screen, we identified a new PBX1 interacting protein containing several zinc finger motifs that we called ZFPIP for Zinc Finger PBX1 Interacting Protein. We demonstrated that ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. We further showed that ZFPIP is able to bind physically and in vivo to PBX1 and moreover, that it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. We suggest that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways.

Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

ZFPIP/Zfp462 is maternally required for proper early Xenopus laevis development

ZFPIP (Zinc Finger Pbx1 Interacting Protein) has been recently identified in our laboratory in a yeast two hybrid screen using an embryonic mouse cDNA library and PBX1 as a bait. This gene encodes a large protein (250 kDa) that contains a bipartite NLS, numerous C2H2 zinc fingers and is highly conserved amongst vertebrates. In order to address the role of ZFPIP during embryonic development, we analysed the expression pattern of the gene and performed morpholinos injections into Xenopus laevis embryos. We first showed that the ZFPIP protein was maternally present in oocytes. Then, ZFPIP was detected from morula to neurula stages in the nucleus of the cells, with a gradient from animal to vegetal pole. By injection of ZFPIP morpholinos, we showed that morphant embryos were unable to undergo proper gastrulation and subsequently exhibited a persistent opened blastopore. Analysis of molecular and cellular events that were altered in morphant embryos highlighted an impairment of cell division processes as illustrated by atypical mitosis with aberrant metaphase, anaphase or telophase, incomplete chromosome segregation or conjointed nuclei. The overall data presented here demonstrated that ZFPIP was a major developing gene that acts in the very first steps of embryonic development of Xenopus laevis.

Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development.

ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N‐cam expression patterns, whereas krox‐20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos‐7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.