P2‐307: A stable G‐quadruplex within the ADAM10 5'‐UTR is involved in translational repression of ADAM10
Alzheimer's & Dementia(2011)
摘要
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.
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关键词
translational repression,g-quadruplex
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