| Alzheimer's Disease (AD) is the most prevalent cause of senile dementia. A large body of evidence implicates beta-amyloid (Abeta) peptide as a prominent causative agent in AD pathophysiology. Abeta is a product of Amyloid Precursor Protein (APP) proteolytic processing. All currently known genetic mutations associated with familial AD result in upregulated APP expression or increased Abeta production. One of the ways APP expression is modulated is through regulation of post-transcriptional stability of APP messenger RNA (mRNA). Cellular APP mRNA levels closely and positively correlate with APP protein levels and downstream Abeta production.;APP mRNA contains several cis-acting regulatory elements that affect its post-transcriptional stability. A 52 base element (52sce), immediately downstream from the stop codon, has been previously shown to complex with uncharacterized cytoplasmic proteins. Affinity purification with 52sce RNA identified six proteins that specifically interact with the 52sce: nucleolin, YB-1, La/SS-B, EF-1alpha, rck/p54 and PAI-RBP1. These proteins co-immunoprecipitate with each other and with APP mRNA from the cytoplasm of cultured human neuroblasts.;One of the 52sce-binding proteins, rck/p54, is a member of the highly conserved DEAD-box family of RNA helicases involved in virtually every stage of mRNA metabolism, including control of RNA stability. Augmentation of intracellular rck/p54 protein levels, either through TAT-protein transduction or through rck/p54 cDNA transfection into live cells, resulted in APP mRNA and protein overexpression comparable to that seen in Down syndrome and AD brain tissue. Deletion of the 52sce markedly diminished the effect of rck/p54 overexpression on eGFP-APP 3'-UTR chimeric mRNA steady-state levels, indicating that rck/p54 stabilizes APP mRNA in a 52sce-dependent manner.;Sequential mutations modulated ex vivo binding affinity of the 52sce to its binding proteins and to RNAse. Mutations that diminished the protein binding affinity and RNAse protection markedly decreased the half-life of APP mRNA in cultured neuronal cell lines. Conversely, mutations that resulted in increased protein-binding affinity and increased RNAse protection of 52sce resulted in a dramatically prolonged half-life of APP mRNA in the cultured cells. These data indicate that interactions between 52sce and its protein cofactors modulate the stabilizing function of the element. |
