P3-335: Upstream of N-ras (UNR) is involved in translational control of ADAM10 protein expression

Abstract

Background: The amyloid beta peptide (A ) is derived by proteolytic processing of the amyloid precursor protein (APP) by the beta-secretase BACE1 and gamma-secretase. In contrast to this amyloidogenic processing, APP is predominantly cleaved by the alpha-secretase within the A domain and this precludes the formation of A . We and other research groups could show that BACE1 protein expression is regulated by the 5’untranslated region (UTR) of the BACE1 mRNA, however little is known about the regulation of alpha-secretase. Similar to the 5’UTR of BACE1, the 5’UTR of ADAM10 consists of 444 nucleotides with a GC-content of 70% and two upstream open reading frames. We hypothesize that ADAM10, the major anti-amyloidogenic alpha-secretase, is also regulated by its 5’UTR and postulate that ADAM10 and BACE1 are regulated on a posttranscriptional level by 5’UTR binding proteins. Methods: We performed a large mutagenesis analysis to identify regions within the 5’UTR of ADAM10 which are important for translational regulation. To identify possible binding candidates we performed a UTR-database search. Results: We found that ADAM10 expression is regulated by its 5’UTR. In the presence of the 5’UTR we observed a significant reduction of ADAM10 protein levels in HEK293 cells. mRNA levels were not affected. Deletions from the 3’ end of the 5’UTR led to an even stronger inhibition of ADAM10 expression. However, a stepwise deletion of the first 259 nucleotides from the 5’end resulted in a strong increase in ADAM10 protein expression. Moreover we show that RNA binding proteins exist which bind specifically and selectively to either the 5’UTR of ADAM10 or BACE1. Using Electophoretic Mobility Shift Assays we identified UNR, a cytosolic RNA binding protein, as one binding partner of the ADAM10 5’UTR. Deleting the two well conserved binding sites within the UTR abolished the binding of recombinant UNR to the 5’UTR of ADAM10 and resulted in an increase of ADAM10 expression after transient transfection. Conclusions: In this sudy we demonstrate that the cytoplasmic protein UNR is able to bind to the 5’UTR of ADAM10 and that UNR might be involved in translational regulation of ADAM10 protein expression.