A novel mRNA decay inhibitor abolish pathophysiological cellular transition

The synthesis and decay of mRNA in cells are carried out by influencing each other, and their balance is altered by either external or internal cues, resulting in changes in the dynamics of the cell. We have previously reported that it is indispensable that an array of mRNAs which shape a phenotype should be 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 a-helix analog with an alkaloid backbone to determine a compound inhibiting the binding between DDX6 and 4E-T proteins, because the interaction is executed between a-helix of structured and internally disordered protein, respectively, and identified IAMC-00192 as a lead compound. The compound showed a direct inhibition in the interaction between DDX6 and 4E-T by using WAVE system with a KD of 774.3 nM. The compound inhibited PB formation that temporally increases during adipogenesis and epithelial-mesenchymal transition (EMT), and significantly suppressed their cellular transition. We also found that in the EMT model, the half-life of pre-existing mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay will be not only a tool to analyze in detail what pathological conditions RNA decay affects and how it regulates the state, but also lead to drug discovery of the first in class as RNA decay inhibitor. Overall design: We established a cell line (A549EGFP-DDX6) in which a protein fused with EGFP and DDX6 was introduced into A549 using a retrovirus. In A549 EGFP-DDX6, the processing body (PB) is fluorescent to EGFP. The cells were stimulated with TGFB2 (Tb2, 5 ng/ml) for 2 days, and PB fractions were isolated from epithelial-mesenchymal transition (EMT)-induced cells by FACS. The isolated PB fractions were subjected to RNA extraction and compared with cytoplasmic fractions. Each RNA-seq result was normalized by FPKM, and the gene with higher expression level than the average expression level of all genes was considered as the expressed gene and compared with each sample.