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Titel: Translational Regulation of mRNAs encoding SAPAP3 in Rattus norvegicus
Sonstige Titel: Regulation der Translation von SAPAP3 mRNA Molekülen in Rattus norvegicus
Sprache: Englisch
Autor*in: Chua, Jia En John
Schlagwörter: postsynaptische Dichte; postsynaptic density
GND-Schlagwörter: Gehirn; Synapse; mRNA; Translation
Erscheinungsdatum: 2006
Tag der mündlichen Prüfung: 2006-05-12
SAPAP3 is one of the four members of the family of synapse-associated protein
90/postsynaptic density-95-associated proteins which act as adaptor proteins in the
assembly of the postsynaptic density (PSD) in neuronal excitatory synapses. Of the
four members, only transcripts belonging to SAPAP3 are dendritically localized.
Using a combination of database searches and RT-PCR, I have isolated three different
SAPAP3 5’ untranslated region (UTR) sequences. One of these variants (E1B variant)
is evolutionarily conserved, long, highly GC-rich and is predicted to form stable
secondary structures. These features strongly suggest its involvement in regulating
translation of its mRNA. Inclusion of the E1B variant 5’ UTR sequence in constructs
encoding SAPAP3 or heterologous proteins leads to a drastic reduction in
recombinant protein expression. Northern analysis revealed that mRNA levels are not
affected by the inclusion of the 5’ UTR indicating that protein expression is inhibited
at the translational level. In vitro competition assays ruled out the involvement of
trans-acting proteins in this process. Sequential deletions of the 5’ UTR segments
indicated that secondary structures are not responsible for translational inhibition
while mutation of the start codon of only one of the four upstream open reading
frames (uORF2) is sufficient to elevate protein synthesis to comparable levels as
observed in the absence of the complete 5’ UTR. Remarkably, the E1B 5’ UTR
sequence leads to the synthesis of two SAPAP3 isoforms when positioned directly in
front of the native SAPAP3 coding region. Synthesis of the shorter SAPAP3b isoform
is achieved via alternative translational initiation from a downstream in-frame start
codon (AUG+277). Initiation from AUG+277 is enhanced by the presence of uORF2
indicating that uORF2 functions as a reinitiation shunt to direct ribosomes away from
the AUG of SAPAP3a. SAPAP3a differs from SAPAP3b by an additional 92 amino
acids at its N-terminus. The ratios of SAPAP3 isoforms vary in distinct brain regions
examined suggesting functional differences between both isoforms. Over-expression
of SAPAP3a in hippocampal neurons indicated strong enrichment of SAPAP3a at
excitatory synapses. While no gross morphological disturbances are observed in these
neurons, changes in glutamatergic transmission resulting from perturbations of
endogenous SAPAP3 isoform ratios may exist which were not examined so far. The
synthesis of two SAPAP3 isoforms via alternative translational initiation distinguishes
SAPAP3 from the other PSD proteins and reveals an additional mechanism for
generating protein diversity in the PSD.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/1407
URN: urn:nbn:de:gbv:18-29674
Dokumenttyp: Dissertation
Betreuer*in: Richter, Dietmar (Prof. Dr.)
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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