Use of intron-based RNA systems to study nuclear and cytoplasmic RNA interference processes in plants

我们先前已经表明，内含子发夹RNA（int-hpRNA）转基因构建体的转录物1）保留在细胞核中，2）低效地加工成小RNA（sRNA），3）有效地触发转基因靶启动子的RNA指导的DNA甲基化（RdDM），但4）未能触发转基因传感器mRNA的转录后基因沉默。在目前的提案中，我们提出了我们的目标是如何继续我们对基于内含子的RNA干扰（RNAi）系统的研究。我们的研究将集中在阐明int-hpRNA介导的基因沉默的基本方面。然而，我们也将评估int-hpRNA构建体作为生物技术应用工具的潜力。通过使用常规的（非内含子）发夹，内源性启动子已被证明是RdDM和转录基因沉默（TGS）的不良靶标。基于int-hpRNA诱导甲基化的显著精确性和效率，很容易推测int-hpRNA将有效地启动内源性启动子的RdDM和TGS。如果是这样的话，基于内含子的RNAi方法将为可遗传的表观遗传基因沉默提供一个强大的工具。此外，我们的目标是利用基于内含子的RNAi系统来研究内含子对RNA介导的沉默的模糊可及性。最后，主要RdDM触发因子（sRNA或更长的RNA）的争议性质将通过分析非小干扰RNA产生int-hpRNA在Dicer样缺陷植物中触发RdDM的能力来检查。

We have previously shown that the transcripts of an intronic hairpin RNA (int-hpRNA) transgene construct 1) were retained in the nucleus, 2) were inefficiently processed into small RNAs (sRNAs), 3) efficiently triggered RNA-directed DNA methylation (RdDM) of a transgenic target-promoter but 4) failed to trigger post-transcriptional gene silencing of a transgenic sensor mRNA. In the current proposal, we present how we aim to continue our research on intron-based RNA interference (RNAi) systems. Our studies will be focused on the elucidation of basic aspects of int-hpRNA-mediated gene silencing. However, we will also evaluate the potential of int-hpRNA constructs as a tool for biotechnological applications. By using conventional (non-intronic) hairpins, endogenous promoters have been shown to be poor targets for RdDM and transcriptional gene silencing (TGS). Based on the remarkable precision and efficiency of methylation induced by the int-hpRNA, it is tempting to speculate that int-hpRNA would efficiently initiate RdDM and TGS of endogenous promoters. If so, the intron-based RNAi approach would provide a powerful tool for heritable epigenetic gene silencing. In addition, we aim to take advantage of the intron-based RNAi system to investigate the ambiguous accessibility of introns against RNA-mediated silencing. Finally, the debatable nature of the primary RdDM-trigger (sRNAs or longer RNAs) will be examined by analysing the capacity of the non-small interfering RNA-producing int-hpRNA to trigger RdDM in Dicer-like-deficient plants.