A stable G-quadruplex within the ADAM10 5'-UTR is involved in translational repression of ADAM10

Alzheimer's disease is the most common form of dementia worldwide. It is characterized by intracellular neurofibrillary tangles and extracellular amyloid plaques. Amyloid plaques consist mainly of the hydrophobic amyloid ß peptide (Aß), which is liberated upon endoproteolytic processing of the amyloid precursor protein (APP) by consecutive cleavages of ß-secretase and ?-secretase. In contrast, cleavage of APP by a-secretase prevents the formation of Aß. ADAM10 (a disintegrin and metalloprotease) was shown to be the best candidate for a-secretase in vivo. ADAM10 has a 5'-untranslated region (5'-UTR), which belongs to the class of long 5'-UTRs which were demonstrated to regulate translation of mRNAs from many regulatory genes. It is 444 nucleotides long, has a GC-content of 70% and contains 2 upstream open reading frames. HEK293 cells were transiently transfected with plasmids encoding ADAM10 with and without 5'-UTR and ADAM10 variants with a truncated or mutated 5'-UTR. Protein expression levels were compared to mRNA levels. Furthermore, we used CD-spectroscopy to identify a stable G-quadruplex motif within the 5'-UTR of ADAM10. Here we show that the 5'-UTR of ADAM10 regulates the rate of ADAM10 translation. In the absence of the 5'-UTR we observed a significant increase of ADAM10 protein levels in HEK293 cells without affecting mRNA levels. We analyzed the effect of several deletion mutants in order to determine regions within the 5'-UTR which influence the translation efficiency of ADAM10. Successive deletion of the first part of the ADAM10 5'-UTR resulted in a significant increase in ADAM10 protein expression, arguing that this part of the 5'-UTR contains inhibitory elements. Using CD-spectroscopy, we identified a G-quadruplex motif, which forms a very stable secondary structure within the first half of the ADAM10 5'-UTR. Mutation of this G-quadruplex motif results in enhanced ADAM10 expression. We provide evidence that a 30 nucleotide long G-rich region within the first portion of the ADAM10 5'UTR is able to form an extremely stable intramolecular G-quadruplex secondary structure which contributes to the inhibitory effect of the 5'-UTR on the translation of ADAM10.