A novel mRNA decay inhibitor abolishes pathophysiological cellular transition

Abstract

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Fig. 1. A high-throughput screening system for the discovery of small-molecule inhibitors related to RNA degradation.

A Design of an RNA degradation inhibitor screening assay. B Binding and inhibition experiments of Nluc and Cluc using luciferase activity assay. C Cytotoxicity (Rluc) and binding inhibition (Fluc/Rluc) assessed when each inhibitor was added. D DMSO, O Opti-MEM.

Fig. 2. Inhibition of PB formation by the small molecule inhibitors.

A Exposure experiments of candidate factors inhibiting RNA degradation to A549 EGFP-DDX6. GFP foci are PB. Nuclei were stained with Hoechst33342. The graph shows the number of intracellular PBs when each small molecule inhibitor was added. B Protein expression analysis of A549 when a small molecule inhibitor IAMC-00192 was added using Western blotting. Each graph shows the result in triplicate. C Structure of IAMC-00192. The molecular model on the right is the 3D structure described by MolView (https://molview.org).

Fig. 3. Protein-protein interaction analysis using Creoptix WAVEsystem and inhibitor candidate molecules.

A Design of proteins for use in measuring protein-protein interactions. B Expression and purification confirmation of the designed protein. The protein was immunoprecipitated with HA antibody and verified by Western blotting. C Measurement of changes in binding strength due to changes in the concentration of CHD_4E-T protein. R Rinsing the sensor with buffer, I Injection of analyte proteins. D Measurement of the change in binding strength when the RNA degradation inhibitor IAMC-00192 is added.

Fig. 4. Inhibition of processing body formation during induction of adipogenic differentiation.

A Experimental design for induction of adipogenic differentiation: DMEM low DMEM low glucose with 10% FBS, AC adipogenic-cocktail (AC) containing insulin, IBMX, and dexamethasone. ICC Immunocytochemistry. WB Western Blotting. ORO Oil Red O. B Immunocytochemical staining using DDX6 antibody. 3T3-L1 cells were fixed and stained on day 4 of adipogenic differentiation. C WB analysis of 3T3-L1 cells on day 4 of stimulation for adipogenic differentiation. The graph shows the expression ratio of each protein corrected by Gapdh protein expression level. Rep replication. D Phase-contrast images (top row) and ORO staining results (middle and bottom rows) of 3T3-L1 cells on day 8 of stimulation for adipogenic differentiation. The middle row is a magnified image, and the lower row is the entire image of well (12-well plate). E Gene expression analysis of 3T3-L1 cells stimulated by the adipogenic differentiation over time by qPCR. Design of the analysis of RNA expression in the cytoplasm and PB.

Fig. 5. Analysis of RNA molecules contained in PB using a fluorescence-activated particle sorting.

A The cells were A549 EGFP-DDX6, in which EGFP-DDX6 was expressed by pMXs retrovirus vector. Each cell was separated into cells with TGFB2 (Tb2) and without TGFB2 (CNT), and the cytoplasmic fraction (Cyto.) of each cell was collected, and RNA was collected. Additionally, EGFP-fluorescent PBs (PB) were isolated from the cytoplasmic fraction by FACS. B Representative figure for PB sorting. C Quality check of RNA collected by PB sorting. The evaluation was done with an Agilent Bioanalyzer (n = 1). D The exploration of gene groups whose gene expression levels in each condition are higher than the average statement value (n = 1). The expression levels were quantified by fragments per kilobase of exon per million reads mapped (FPKM). E Venn diagram for each condition. F Gene Ontology analysis results using DAIVD for each condition.

Fig. 6. Inhibition of epithelial-mesenchymal transition by a small molecule inhibitor.

A Phase-contrast microscopy image and fluorescence image stained with phalloidin-FITC of EMT differentiated A549 cells with Tb2 for 2 days. B Tb2 was added to A549 cells for 2 days, and cells stained with phalloidin-FITC were analyzed by FACS. Results of quantitative analysis of EMT-differentiated A549 stained with phalloidin-FITC by FACS. The left figure shows the FACS results, and the right graph shows the FITC fluorescence intensity of these results quantified by the geometric mean. geoMFI geometric mean fluorescence intensity. C Gene expression analysis in relation to EMT using the qPCR method. D Inhibition analysis of RNA degradation in the early stage of the induction of IAMC-00192.