Doctoral thesis (Dissertations and theses)
étude fonctionnelle des facteurs d’épissage SR (Ser/Arg-rich) au cours du développement embryonnaire de Danio rerio.
Joris, Marine
2017
 

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Keywords :
Splicing; SR proteins; Morpholinos
Abstract :
[en] Ser/Arg-rich (SR) proteins constitute a phylogenetically conserved family of RNA binding proteins characterized by one or two RNA-recognition motifs (RRMs) at the N-terminus and a C-terminal domain enriched in Ser/Arg dipeptides. They are important regulators of constitutive and alternative splicing. Moreover, SR proteins are involved in many other aspects of RNA metabolism, including transcription, non-sense mediated decay, mRNA export, translational control as well as maintenance of genome stability. While the roles of SR splicing factors have been widely studied at a molecular level, their functions during animal cell differentiation and development are still largely undetermined. In this study, we decided to take advantage of the vertebrate model organism Danio rerio (zebrafish) to investigate SR protein functions by using molecular and genetic approaches. Fifteen zebrafish SR genes were identified belonging to the three subfamilies of SR proteins [SRSF1 (1RRM and 1 pseudo-RRM), SRSF2 (1RRM) and SRSF7 (1RRM +1ZnK)] initially described in humans. The current PhD thesis aimed to study of four of them, Srsfs5a, Srsf9, Srsfs7 and Srsf2b. We first determined the expression pattern of all SR genes by in situ hybridization at 24, 48 and 72 hours post-fertilization. This analysis revealed an ubiquitous expression pattern for SR genes, however pointing a higher expression in the brain and in the eyes. We next performed “knock-down” experiments using morpholinos (MOs) microinjection to determine SR gene functions. One splicing MO, designed to target srsf5a (sMOsrsf5a) showed an interesting phenotype that coincides with the expression pattern of the gene in the eyes. As none of the four other MOs used to target srsf5a could confirm the phenotype, we generated stable knockout mutant lines using TALEN and CRISPR/Cas9. Interestingly, the corresponding homozygous mutants did not display any phenotypic traits. These inconsistencies were attributed to a possible genetic compensation mechanism in mutants, but also to an underestimated large number of inadvertent morpholino RNA targets. Indeed, we uncovered that only 11 consecutive bases complementary to the 25 MO bases are sufficient for binding and subsequent blocking of splice sites, suggesting that the sMOsrsf5a induced phenotype was not due to the inactivation of this unique, specific gene. In addition, we observed that sMOsrsf5a secondary targets can be slightly reduced by increasing embryos growth temperature after microinjection. Our study contributes to the debate concerning MO specificity, efficacy and the number of unknown targeted sequences.  
Research center :
Génomique fonctionnelle et imagerie moléculaire végétale
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Joris, Marine ;  Université de Liège - ULiège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
Language :
French
Title :
étude fonctionnelle des facteurs d’épissage SR (Ser/Arg-rich) au cours du développement embryonnaire de Danio rerio.
Alternative titles :
[en] Functional study of Ser/Arg-rich splicing factors during zebrafish embryonic development.
Defense date :
05 September 2017
Number of pages :
284
Institution :
ULiège - Université de Liège
Degree :
Doctorat en Sciences, orientation Biochimie, Biologie moléculaire et cellulaire, Bioinformatique et modélisation
Promotor :
Motte, Patrick ;  Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
Muller, Marc  ;  Université de Liège - ULiège > GIGA > GIGA I3 - Laboratory for Organogenesis and Regeneration
President :
Remacle, Claire  ;  Université de Liège - ULiège > Integrative Biological Sciences (InBioS)
Secretary :
Peers, Bernard ;  Université de Liège - ULiège > GIGA > GIGA Stem Cells - Zebrafish Development and Disease Model
Jury member :
Dequiedt, Franck  ;  Université de Liège - ULiège > GIGA > GIGA Molecular Biology of Diseases
Rezsohazy, René
Stainier, Didier
Name of the research project :
étude fonctionnelle des facteurs d’épissage SR (Ser/Arg-rich) au cours du développement embryonnaire de Danio rerio.
Funders :
FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
Commentary :
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Available on ORBi :
since 02 October 2017

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