Related Experiment Videos
Multiple proteins interact at a unique cis-element in the 3'-untranslated region of amyloid precursor protein mRNA
S H Zaidi1, R Denman, J S Malter
1Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53792-2472.
Abstract:
Growing evidence suggests that Alzheimer's disease results from dysregulated production and deposition of beta-amyloid in the central nervous system. beta-Amyloid is derived from proteolytic processing of one of multiple amyloid precursor protein (APP) isoforms. The production of APP in many somatic tissues and tumor cell lines provides a more accessible model to study the regulation of APP gene expression. Recent data suggest that APP mRNAs accumulate in activated lymphocytes and neuronal tumor lines. We are interested in defining the contribution of alterations in stability to changes in steady-state APP mRNA levels in these model systems. Herein we demonstrate by mobility shift assay that the 3'-untranslated region of APP RNAs which contain a contiguous 29-base region interacts in vitro with multiple mRNA-binding proteins found in cytosolic lysates prepared from normal and transformed human cells. UV cross-linking of radiolabeled APP RNAs to cytosolic protein extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified six distinct RNA-protein complexes of 42, 47, 65, 73, 84, and 104 kDa. Competition assays with APP, AU-rich, or irrelevant RNAs demonstrated that binding was specific and in some cases preferential for AU- or U-rich sequences by which we tentatively place the binding site of the proteins along the 29-base region. APP mRNA-binding proteins were constitutively active in all tumor lines examined as well as at diminished levels in whole human brain cytosolic lysates. The core element is AU-rich and highly conserved between human and some murine APP mRNAs. In the accompanying paper (Zaidi, S. H. E. and Malter, J. S. (1994) J. Biol. Chem. 269, 24007-24013) we show that this 29-base element in the 3'-untranslated region regulates the stability of APP mRNA. Cumulatively these data suggest that steady-state APP mRNA levels are modulated by cytosolic protein-RNA interactions.
Insights
Alzheimer's disease may involve amyloid precursor protein (APP) mRNA regulation. Researchers found specific proteins bind to APP mRNA's 3' untranslated region, influencing its stability and levels in cells and brain tissue.
Area of Science:
- Molecular Biology
- Neuroscience
- Biochemistry
Background:
- Alzheimer's disease is linked to amyloid precursor protein (APP) dysregulation.
- APP mRNA accumulation occurs in activated lymphocytes and neuronal tumor lines.
- Understanding APP mRNA stability regulation is crucial for studying its gene expression.
Purpose of the Study:
- To investigate the role of mRNA stability in regulating steady-state APP mRNA levels.
- To identify proteins that bind to APP mRNA and characterize their interactions.
- To determine if these interactions are specific and conserved.
Main Methods:
- Mobility shift assays to detect RNA-protein interactions.
- UV cross-linking and SDS-PAGE to identify and characterize RNA-binding proteins.
- Competition assays to assess binding specificity and sequence preference.
Main Results:
- A 29-base region in the APP mRNA 3'-untranslated region binds multiple cytosolic proteins.
- Six distinct RNA-protein complexes were identified, with sizes ranging from 42 to 104 kDa.
- Binding was specific and showed preference for AU- or U-rich sequences, conserved between human and murine APP mRNA.
Conclusions:
- Cytosolic proteins bind to a specific region of APP mRNA.
- These protein-RNA interactions modulate steady-state APP mRNA levels.
- This mechanism is relevant in both tumor cell lines and human brain tissue.