13 research outputs found
Zebrafish as a Model to study Human Disease: Functional Studies of the FXR Proteins
The FMR1 protein, FMRP, has been extensively studied due to its involvement in the fragile
X syndrome, which is mainly characterised by mental retardation, macroorchidism and facial
dysmorphologies. The FMR1 gene is transcriptionally inactivated by the methylation of its
promoter region due to the expansion of a CGG repeat. Thus the fragile X syndrome is caused
by the absence of the protein FMRP. FMRP is part of a small RNA binding protein family,
which also include FXR1P and FXR2P. Due to the high homology of important functional
domains and similar expression patterns in the brain, FXR1P and FXR2P are thought to have a
similar function to FMRP. All three proteins are ubiquitously expressed with high expression in
brain and testis. In addition, FXR1P is highly expressed in striated muscle tissue. The cellular
function of the FXR proteins has been studied by the generation of knockout mouse models
for all three genes. The Fmr1 KO mice display similar features as fragile X patients, including
learning and memory abnormalities and macroorchidism. Further characterisation of the Fmr1
KO mice showed a reduced pruning and/or maturation of spines, which normally occurs during
late embryonic and postnatal development. Fxr1 KO mice showed a severe striated muscle
phenotype and died shortly after birth. This early lethality indicates that cellular Fxr1p function
is critical during embryonic development. Fxr2 KO mice showed a discrete behavioural
phenotype, but needs further characterisation to determine whether Fxr2p has a role during
embryonic development. Processes during embryonic development are difficult to study in the
mouse model due to in utero development of the embryos. For this reason, we have chosen
the zebrafish (Danio rerio) as a model to study the FXR protein function especially during
embryonic development.
The zebrafish is a well-established simple vertebrate animal model. Their ability to be kept in
large numbers and the ease of breeding make them easy to maintain. Due to its transparent
embryos that develop externally the zebrafish represents an ideal model to study processes
during embryonic development. In addition, the embryos develop quickly from a single cell to
something that is recognizable as a fish after 24 hours of development. The favourite method
to study gene function in zebrafish is the generation of a morpholino-mediated knockdown
zebrafish. This morpholino technology is based on the injection of synthetic antisense
oligonucleotides into a 1-8 cell stage embryo, which subsequently binds to the mRNA molecule
and thereby preventing translation. The introduction of this thesis (chapter 1) describes the use
of zebrafish as a vertebrate animal model with special emphasis to human disease (part 1) and
introduces the FXR protein family with its three individual members (part 2). The aim of this
thesis is to gain insight in the function of the FXR proteins, and their role during embryonic
development particularly using the zebrafish as a model system
Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2
Expansion of the CGG trinucleotide repeat in the 5′-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs are locally translated in response to stimuli.Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 Å, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9.The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner