The gene repression at the translation stage appears to be a waste of energy for cells
The gene repression at the translation stage appears to be a waste of energy for cells. gene in cells, indicating that the RNA G-quadruplex plays a key role in translational regulation (34,37). To understand Rabbit polyclonal to FBXW12 the properties and biological roles of RNA G-quadruplexes in cells, several small-molecule tools selective for RNA G-quadruplexes have been developed (33,38C50). For example, anthrafurandione and anthrathiophenedione molecules bind to RNA G-quadruplexes in the 5-UTR of oncogene transcript to impair its translation with concomitant anti-cancer properties, exemplifying that RNA G-quadruplexes in oncogene transcripts may serve as drug targets (47). Despite these efforts, specific exogenous control over the cellular roles of RNA G-quadruplexes with a small molecule remain challenging. We previously reported a selective small-molecule RNA G-quadruplex stabilizer, RGB-1, which is capable of repressing the endogenous protein synthesis from RNA G-quadruplex-containing mRNAs in mammalian cells (37). The translational inhibition activity of RGB-1 led us to discover another RNA G-quadruplex latent in the 5UTR of a proto-oncogene mRNA, raising the possibility that RGB-1 serves as a valuable chemical tool for exploration of functional CGP-52411 G-quadruplex-forming sites in a large collection of mRNAs. In the present study, we complement and advance this past work by developing an RGB-1-based approach to search for functional RNA. G-quadruplexes located in the 5UTR region. The approach described in the present study could pave the way for further discoveries of RNA CGP-52411 G-quadruplexes and subsequently a CGP-52411 further understanding of the cellular roles of RNA G-quadruplex. MATERIALS AND METHODS RGB-1 The G-quadruplex stabilizing ligand used in the present study, RGB-1, was originally discovered by chemical library screening in our own laboratory, and its structure was confirmed by chemical synthesis as described in our previous publication (37). The ligand used in the present study was also synthesized in house as described before (37). Solubility determination The solubility of RGB-1 was determined by UV absorption analysis. 1.196, 0.597?and 0.598 mg of RGB-1 were dissolved in 100 l of DMSO. The UV absorbance of each RGB-1 solution was analyzed by a UV-1650PC (SHIMADZU). of the solution. The maximal solubilities of CGP-52411 RGB-1 in 10% and 1% DMSO/H2O were 26 and 11 M, respectively. RNA preparation RNA samples for UV and CD measurements and footprinting assay were solubilized in 10 mM TrisCHCl (pH 7.5) buffer containing 100 mM KCl or LiCl. Prior to use, the samples were heated to 90C for 5 min and gradually cooled to room temperature over 60 min in the same buffer. Proteome profiler array The Human XL CGP-52411 Oncology Array Kit (R&D Systems) was used for the parallel determination of relative levels of 84 human cancer-related proteins. MCF7 cells were maintained in medium A (Dulbecco’s modified Eagle’s medium, supplemented with 100 units/ml penicillin, 100 g/ml streptomycin sulfate, and 10% (v/v) fetal bovine serum) at 37C in a humidified 5% CO2 incubator. On Day 0, MCF7 cells were added to medium A in a 35 mm dish at 4.0??105 cells per well. On Day 1, the cells were treated with 1% DMSO (control) or 10 M RGB-1 in 1% DMSO. After 2 days of culture, the cells were washed three times with cold Phosphate-Buffered Saline (PBS), and lysed with buffer 17 (# 895943, R&D Systems). Blotting assays were performed according to the manufacturer’s protocol as follows. Array membranes were treated with 2.0 ml of Array Buffer 6 for 1 h?for blocking. After blocking, the membranes were incubated with the cell lysates overnight at 4C on a rocking platform shaker. Each membrane was washed with 1?Wash Buffer for 10 min?for a total of three washes. The membranes were treated with 1.5 ml of Detection Antibody Cocktail and incubated for 1 h. The membranes were treated with 2.0 ml of 1 1?streptavidinCHRP and Incubated at room temperature for 30 min, and then washed each membrane. After washing, the membranes were treated with 1.0 ml of the Chemi Reagent Mix. and incubated for 1 min. After squeezing out the excess Chemi Reagent Mix, the membranes were placed in an autoradiography film cassette with the identification numbers facing up, and the X-ray film was exposed for 10 min?for protein expression analysis. Western blot analysis MCF7 cells were maintained in medium A (Dulbecco’s modified Eagle’s medium, supplemented with 100 units/ml penicillin, 100 g/ml streptomycin sulfate, and 10% (v/v) fetal bovine serum) at 37C in a humidified 5% CO2 incubator. On Day 0, MCF-7 cells were added to medium A in a six-well plate at 3??106 cells per well. On Day 1, the cells were treated with 1%.