Post-transcriptional control of gene expression : therapeutic targeting in human diseases

SUMMARY RNA has become a promising target for pharmacological purposes. In this study, evidence are provided of the up-regulation of messenger RNA in a sequence-specific manner by targeting a destabilizing element. Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE Binding Proteins (AUBPs) that are involved in the decay process. The bcl-2 (b)-ARE, located in the 3 -UTR of the b-RNA, regulates the rate of bcl-2 RNA degradation. The b-ARE has been targeted with three 2 -O-methyl oligoribonucleotides designed in the antisense orientation (asORNs) and, afterwards, with three 2 -O-Me-ORNs, homologous to the core region of ARE of bcl-2 mRNA. Both asORNs and sense-ORNs were studied by a cell-free degradation assay. ORNs inhibited the rate of RNA decay in a dose-response and ORNs sequence dependent fashion, moreover asORNs were specific to the b-ARE. ORNs were then transfected into malignant cells in culture and b-RNA half-life was measured by real-time PCR. We showed that ORNs increased the expression of b-RNA and Bcl-2 protein in a dose-response fashion, while asORNs were Bcl-2 specific and affected also cell survival and phenotype, sORNs up-regulated the expression ARE-transcripts other than bcl-2. In fact, sORNs, tested for decoy-aptamer activity in UVx-linking and RNA-IP assays, competed with the ARE motif for the interaction with AUBPs shared by different ARE-transcript, hence their activity is bcl-2 independent.