Translational Regulation of mRNAs encoding SAPAP3 in Rattus norvegicus

Abstract

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.