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See detailFunctional study of the Ser/Arg-rich splicing factor SRSF5a during zebrafish embryonic development
Joris, Marine ULg; Muller, Marc ULg; Motte, Patrick ULg

Poster (2014, June 05)

Nuclear pre-mRNA splicing is a key process regulating gene expression in eukaryotes. Splicing consists in the removal of introns and the joining of exons within a dynamic macromolecular complex called the ... [more ▼]

Nuclear pre-mRNA splicing is a key process regulating gene expression in eukaryotes. Splicing consists in the removal of introns and the joining of exons within a dynamic macromolecular complex called the spliceosome, which consist of five small nuclear ribonucleoproteins (snRNPs) and numerous non snRNPs proteins (1). Amongst these non snRNPs proteins, the SR proteins family constituted an important group of splicing factors that are involved in constitutive and alternative splicing (2,3). SR proteins are structurally related as they are characterized by one or two RNA-recognition motifs (RRMs) in N-ter and a C-terminal domain enriched in dipeptide Ser/Arg. Phylogenetic inference using the RRM domain allowed us to identify 13 encoding genes for SR proteins in the vertebrate model organism, Danio rerio. The Zebrafish is increasingly recognized as a powerful model for the study of vertebrate embryonic development in a physiological context. The roles of SR splicing factors during animal cell differentiation and development are largely unknown. The aim of the present research is to investigate SR proteins functions during zebrafish development by using molecular and genetic approaches. In this study, we investigated the role of the SR splicing factor SRSF5a. The expression profile was determined by in situ hybridization at 24, 48 and 72 hours post-fertilization and showed SRSF5a expression mainly in brain, retina and pharyngeal arches at these stages. Furthermore, SRSF5a knock-down by morpholinos microinjection strongly suggests an important role of this specific splicing factor during eyes and brain development. In order to gain insight into the molecular function of SRSF5a, we analysed control and morphant transcriptomes using high throughput RNA sequencing. Finally, we use a complementary approach to morpholinos and generate SRSF5a mutant fishes using TALENs (Tal effector nucleases)(4,5). 1. Wahl MC, Will CL, & Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136(4):701-718. 2. Long JC & Caceres JF (2009) The SR protein family of splicing factors: master regulators of gene expression. Biochem J 417(1):15-27. 3. Graveley BR (2000) Sorting out the complexity of SR protein functions. RNA 6(9):1197-1211. 4. Moore et al. (2012) Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs). Plos One 120(1):1-12. 5. Cade et al (2012) Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic acid research Vol 40, No 16:8001-8010. [less ▲]

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See detailComplex regulation of the FRD3 gene in Arabidopsis relatives
Charlier, Jean Benoit; Polese, Catherine ULg; Nouet, Cécile ULg et al

Poster (2014, March 31)

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See detailFunctional study of the Ser/Arg-rich splicing factor SRSF5a during zebrafish embryonic development.
Joris, Marine ULg; Larbuisson, Arnaud ULg; Muller, Marc ULg et al

Poster (2013, April 18)

To investigate the role of the splicing factor SRSF5a during zebrafish embryonic development, we performed SRSF5a knockdown by morpholino microinjection and we analysed control and morphant transcriptomes ... [more ▼]

To investigate the role of the splicing factor SRSF5a during zebrafish embryonic development, we performed SRSF5a knockdown by morpholino microinjection and we analysed control and morphant transcriptomes using RNA sequencing. [less ▲]

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See detailComplex regulation of the FRD3 gene in Arabidopsis relatives
Charlier, Jean-Benoit ULg; Polese, Catherine ULg; Nouet, Cécile ULg et al

Poster (2013, February 26)

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See detailHard Selective Sweep and Ectopic Gene Conversion in a Gene Cluster Affording Environmental Adaptation
Hanikenne, Marc ULg; Kroymann, Juergen; Trampczynska, Aleksandra et al

in PLoS Genetics (2013), 9

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See detailUse of red autofluorescence for monitoring prodiginine biosynthesis
Tenconi, Elodie ULg; Guichard, Paul; Motte, Patrick ULg et al

in Journal of Microbiological Methods (2013), 93

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See detailInhibition of Tumor Angiogenesis and Growth by a Small-Molecule Multi-FGF Receptor Blocker with Allosteric Properties.
Bono, Francoise; De Smet, Frederik; Herbert, Corentin et al

in Cancer Cell (2013), 23(4), 477-88

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the ... [more ▼]

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment. [less ▲]

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See detailPromoter analysis of three HMA4 gene copies in the zinc hyperaccumulator Arabidopsis halleri
Nouet, Cécile ULg; Cebula, Justyna; Motte, Patrick ULg et al

Poster (2012, September 20)

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See detailZinc hyperaccumulation: a model to examine metal homeostasis in plants
Hanikenne, Marc ULg; Nouet, Cécile ULg; Charlier, Jean-Benoit et al

Conference (2012, September 20)

The plant Arabidopsis halleri exhibits naturally selected metal hypertolerance and extraordinarily high levels of leaf metal accumulation. Metal hyperaccumulator species attract interest as they represent ... [more ▼]

The plant Arabidopsis halleri exhibits naturally selected metal hypertolerance and extraordinarily high levels of leaf metal accumulation. Metal hyperaccumulator species attract interest as they represent an extreme end of natural variation of the metal homeostasis network. This might be useful to reveal the global functioning of metal homeostasis networks and uncover key nodes whose alterations can drastically modify metal accumulation and tolerance. In addition, metal hyperaccumulation is a compelling model to study adaptation. In the last few years, major progress has been achieved in our understanding of the mechanisms underlying metal hyperaccumulation in A. halleri. High rates of metal uptake by roots, root-to-shoot translocation and efficient shoot vacuolar sequestration play central roles in determining hyperaccumulation and hypertolerance. Enhanced functions of several metal transporter-encoding genes result from gene copy number amplification and/or (cis)-regulatory changes. We will describe the function of several transporters in zinc and cadmium hyperaccumulation and hypertolerance, and in the adjustment of nutrient homeostasis in A. halleri. Recent work aiming to determine if selection acted during the evolutionary history of A. halleri on a loci required for metal tolerance and accumulation will be presented. [less ▲]

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See detailFunctional study of Arabidopsis thaliana ASF/SF2-like pre-mRNA SR splicing factors
Stankovic, Nancy ULg; Tillemans, Vinciane; Leponce, Isabelle et al

Poster (2012, July 04)

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See detailEvolution of Metal Hyperaccumulation in Arabidopsis halleri
Hanikenne, Marc ULg; Kroymann, Juergen; Bernal, Maria et al

Conference (2012, February 06)

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See detailA Single Ancient Origin for Prototypical Serine/Arginine-Rich Splicing Factors1[W][OA]
Califice, Sophie; Baurain, Denis ULg; Hanikenne, Marc ULg et al

in Plant Physiology (2012), 158(2), 546-560

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have ... [more ▼]

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have been suggested to be crucial in plant-specific forms of developmental regulation and environmental adaptation. Despite their functional importance, little is known about their origin and evolutionary history. SR splicing factors have a modular organization featuring at least one RRM domain and a C-terminal region enriched in Ser/Arg dipeptides. To investigate the evolution of SR proteins, we infer phylogenies for >12,000 RRM domains representing >200 broadly sampled organisms. Our analyses reveal that the RRM domain is not restricted to eukaryotes and that all prototypical SR proteins share a single ancient origin, including the plant-specific SR45 protein. Based on these findings, we propose a scenario for their diversification into four natural families, each corresponding to a main SR architecture, and a dozen subfamilies, of which we profile both sequence conservation and composition. Finally, using operational criteria for computational discovery and classification, we catalogue SR proteins in 20 model organisms, with a focus on green algae and land plants. Altogether, our study confirms the homogeneity and antiquity of SR splicing factors, while establishing robust phylogenetic relationships between animal and plant proteins, which should enable functional analyses of lesser characterized SR family members, especially in green plants. [less ▲]

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See detailReconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.
Larosa, Véronique ULg; Coosemans, Nadine ULg; Motte, Patrick ULg et al

in Plant Journal (The) (2012), 70

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate ... [more ▼]

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which could hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (1) respiratory-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (2) transformation of the mitochondrial genome is feasible, (3) Chlamydomonas complex I is close to that of humans. To illustrate that, we have introduced a Leu157Pro substitution in the Chlamydomonas ND4 subunit of complex I of two different recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defect when it is present in the heteroplasmic state in a patient presenting chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent the assembly of the 950 kDa whole complex I which conserves nearly all the NADH dehydrogenase activity of the peripheral arm. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect ubiquinone fixation to the membrane domain. The in vitro defects are correlated in vivo with a decrease in dark respiration and growth rate. [less ▲]

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See detailThe HMG-Box Transcription Factor Sox4b Is Required for Pituitary Expression of gata2a and Specification of Thyrotrope and Gonadotrope Cells in Zebrafish.
Quiroz O'Donova, Yobhana ULg; Lopez, Mauricio; Mavropoulos, A et al

in Molecular Endocrinology (2012), 26(6), 1014-1027

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and ... [more ▼]

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshbeta), glycoprotein subunit alpha (gsualpha), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshbeta and gsualpha expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slbeta, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshbeta and gsualpha expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshbeta and gsualpha expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhbeta and fshbeta) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary. [less ▲]

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See detailRUNX3, EGR1 AND SOX9B FORM A REGULATORY CASCADE REQUIRED TO MODULATE BMP-SIGNALING DURING CRANIAL CARTILAGE DEVELOPMENT IN ZEBRAFISH.
Dalcq, Julia ULg; Pasque, Vincent; Ghaye, Aurélie ULg et al

in PLoS ONE (2012), in press

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions ... [more ▼]

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish. [less ▲]

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