Unveiling the molecular mechanism of gene regulation by single molecule approaches

조회수 : 520 등록일 : 2018.11.16 10:27

일시 : 2018.11.14 17:00
소속 : 중앙대학교 화학과
발표자 : 고혜란
장소 : AS510

  RNA interference (RNAi) is a gene regulation pathway induced by small RNA molecules, microRNA (miRNA) and small interfering RNA (siRNA). It is a multi-step process consisting of the small RNA genesis by Dicer/TRBP, guide strand selection and the guide strand-mediated gene silencing by RNA-induced silencing complex (RISC). Despite of the well characterized RNAi pathway, it still remains unclear how the structural features of small RNAs contribute to dicing and subsequent gene silencing efficiency. Here, we incorporated single-molecule fluorescence resonance energy transfer (smFRET) to investigate the molecular interaction between RNA and the components of RISC and discovered that TRBP diffuses specifically on dsRNA, which contributed to enhancing miRNA and siRNA processing by Dicer. Also, with single-molecule fluorescence in situ hybridization (smFISH), we could count the number of mRNAs at single-cell level and quantified the RNAi efficiency by screening various structures of small RNAs. We found that dicing rate and silencing efficiency both increase as a function of the loop length of the small RNAs in a correlated manner. In contrast, mismatches in the stem drastically diminish the silencing efficiency without impacting the dicing rate. Our results imply that the stem structures prevalent in cellular miRNAs are intended for suboptimal silencing efficiency. The quantitative analysis of RNAi using smFISH together with smFRET would be a powerful platform to study RNAi and other gene-regulating system.

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