Funktionelle Charakterisierung von FMRP, dem Krankheitsgenprodukt des Fragilen X-Syndroms

Fragile X syndrome is the most frequent form of heritable mental
retardation. In the majority of cases the disease is caused by
transcriptional silencing of the FMR1 gene in response to the
expansion of CGG repeats in the 5´UTR of the gene, leading to a
lack of fragile X mental retardation protein (FMRP). Only a few
patients exhibit missense-mutations in the coding region of FMR1
leading to an aberrant gene product. However, little is known about
how FMRP deficiency or malfunction leads to the pathophysiology of
Fragile X syndrome. FMRP is an RNA binding protein that associates
with translating polyribosomes as part of a large messenger
ribonucleoprotein (mRNP). In the present study, it was investigated
whether the protein can act as a regulator of translation. Using
recombinant FMRP, it was shown that the protein suppresses
translation of different mRNAs in rabbit reticulocyte lysate.
Interestingly, FMRP containing an amino acid substitution at
position 304 (FMRPI304N), originally identified in a severely
affected patient, renders the protein incapable of interfering with
translation. In vitro binding experiments revealed that FMRPI304N,
in contrast to wildtype FMRP, was incapable of forming
homo-oligomers. However, its affinity for mRNA was not altered.
These results suggest that oligomerization is a prerequisite for
the function of FMRP in translational inhibition. In order to
identify the protein region responsible for homo- and
hetero-oligomerization with the two autosomal homologues FXR1 and
FXR2, an array of FMRP deletion mutants were tested for
oligomerization properties. In vitro binding experiments showed
that a putative coiled-coil domain (aminoacids 112 to 215) was
required for mutual interaction of all three proteins. Finally, it
was examined whether FMRP is post-translationally modified and
whether this may play a role in the function of this protein. It
was initially demonstrated that both, mammalian cell extract and
casein-kinase II, are capable of FMRP phosphorylation in vitro.
Furthermore phospho-aminoacid analysis of immunoprecipitated human
FMRP revealed phosphorylation of serine residues. Additionally, the
protein can be methylated in vitro in the C-terminal part. The
outlined experimental strategy may be utilized as a tool for the
identification and characterization of FMRP-modifying enzymes. The
obtained data identify FMRP as a negative regulator of translation
and suggest that misregulation of translation of specific mRNAs
lead to the disease phenotype. Analysis of the biogenesis of
spliceosomal U-snRNP was the second project of the Ph.D. thesis.
This process involves the transient export of nuclear encoded U
snRNA to the cytoplasm, the assembly with a set of spliceosomal
proteins (termed Sm-proteins B/B’, D1, D2, D3, E, F, and G) and the
nuclear import of the assembled particle to the nucleus. The
assembly reaction of U-snRNP, although a spontaneous process in
vitro, is facilitated in vivo by a large number of proteins,
including SMN, the protein mutated in the neuromuscular disorder
spinal muscular atrophy. These factors are organised in two
functional units, termed PRMT5-complex and SMN-complex, that
successively cooperate in the assembly reaction. In a first step,
the PRMT5-complex sequesters newly synthesized Sm proteins and
converts arginines in SmB, D1 and D3 to symmetrical
dimethylarginines (sDMA). This enhances their transfer to the
SMN-complex, which facilitates the assembly reaction with the U
snRNA. Whereas in vitro methylation experiments have identified
PRMT5 as the catalytic component of the PRMT5 complex, the
functions of the two known cofactors of this enzyme, pICln and
WD45, were unknown. Using a biochemical approach, an interaction
map of the PRMT5 complex has been established. These studies
revealed that oligomeric PRMT5 directly interacts with pICln and
WD45 via distinct domains. Interestingly, in vitro methylation
experiments further indicated that both cofactors strongly activate
the catalytic activity of the methyltransferase. The transfer of
modified Sm proteins to the SMN-complex was studied in a newly
established assay system. Sm proteins form stable heterooligomeric
complexes composed of SmB/D3, SmD1/D2 and SmE/F/G and it is
believed that these complexes are intermediates in the assembly
reaction. Quite unexpectedly, the studies presented in this thesis
indicate that Sm proteins bind initially as monomers to the
PRMT5-complex to allow for the efficient methylation and transfer
to the SMN complex. The data suggest that hetero oligomerization
takes place in a late step of the assembly reaction, possibly at
the SMN complex prior to U snRNP assembly.