Translational repression of the disintegrin and metalloprotease ADAM10 by a stable G-quadruplex secondary structure in its 5'-untranslated region.

Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-ß peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.

Expression of the anti-amyloidogenic secretase ADAM10 is suppressed by its 5'-untranslated region.

Proteolytic processing of the amyloid precursor protein by alpha-secretase prevents formation of the amyloid beta-peptide (Abeta), which is the main constituent of amyloid plaques in brains of Alzheimer disease (AD) patients. alpha-Secretase activity is decreased in AD, and overexpression of the alpha-secretase ADAM10 (a disintegrin and metalloprotease 10) in an AD animal model prevents amyloid pathology. ADAM10 has a 444-nucleotide-long, very GC-rich 5'-untranslated region (5'-UTR) with two upstream open reading frames. Because similar properties of 5'-UTRs are found in transcripts of many genes, which are regulated by translational control mechanisms, we asked whether ADAM10 expression is translationally controlled by its 5'-UTR. We demonstrate that the 5'-UTR of ADAM10 represses the rate of ADAM10 translation. In the absence of the 5'-UTR, we observed a significant increase of ADAM10 protein levels in HEK293 cells, whereas mRNA levels were not changed. Moreover, the 5'-UTR of ADAM10 inhibits translation of a luciferase reporter in an in vitro transcription/translation assay. Successive deletion of the first half of the ADAM10 5'-UTR revealed a striking increase in ADAM10 protein expression in HEK293 cells, suggesting that this part of the 5'-UTR contains inhibitory elements for translation. Moreover, we detect an enhanced alpha-secretase activity and consequently reduced Abeta levels in the conditioned medium of HEK293 cells expressing both amyloid precursor protein and a 5'-UTR-ADAM10 deletion construct lacking the first half of the 5'-UTR. Thus, we provide evidence that the 5'-UTR of ADAM10 may have an important role for post-transcriptional regulation of ADAM10 expression and consequently Abeta production.