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Translational Regulation of mRNAs encoding SAPAP3 in Rattus norvegicus

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.