Re-evaluating Mechanisms of RNA-mediated Initiation of Transposable Element Silencing in Plants

转座因子是基因组中的DNA片段，可以通过插入新位置来动员和产生突变。为了防止这种致突变的结果，细胞已经进化出干扰转座因子动员的机制。先前的工作建立了小RNA在转座因子抑制中的作用。该项目将在模式植物拟南芥中使用现代基因组学支持的方法，对导致流行机制观点的假设和结论进行“严格测试”，并扩大对转座因子沉默如何触发的理解。 由于转座因子广泛存在于植物和动物的基因组中，因此这些结果有望在调控生物学领域产生广泛的影响。该项目也将有很强的教育影响，因为它强调在三个方面的数据再现性和科学道德：课程丰富的本科生谁将研究整合在他们的实验室课程;每年的推广研讨会为7年级的少数民族学生专注于科学，技术，工程和数学;和直接参与研究的早期职业科学家在博士后，研究生和本科生水平。 转座因子的迁移性和致突变潜力取决于由转座因子自身合成RNA（转录）产生的mRNA。该转录步骤是调节转座因子移动性的关键控制点。先前的工作表明，转座因子的转录被小RNA关闭，小RNA通过转录后干扰机制启动沉默，该机制转变为转录水平沉默。该项目将重现以前工作的关键方面，并将应用现代技术能力来产生更高价值的数据集，这些数据集将以创造性和创新的方式进行分析，以精确确定RNA干扰如何针对特定转录本，以及RNA指导的DNA甲基化和转录水平抑制如何建立，以产生转座因子的跨代表观遗传沉默。数据和协议将通过开放获取途径广泛传播，以确保研究的可重复性。这些结果不仅可以为RNA介导的沉默如何发生提供新的理解，还可以为如何防止这种沉默提供线索。这些见解可能对具有农艺效益的基因工程植物产生实际影响。

Transposable elements are DNA fragments in a genome that can mobilize and create mutations by inserting into new locations. To guard against this mutagenic outcome, cells have evolved mechanisms to interfere with transposable element mobilization. Previous work established a role for small RNAs in transposable element inhibition. This project will use modern genomics-enabled approaches in the model plant, Arabidopsis thaliana, to conduct a 'severe test' of the assumptions and conclusions that led to the prevailing mechanistic view and to expand understanding of how transposable element silencing is triggered. Because transposable elements are widespread in the genomes of both plants and animals, the results are expected to have broad impact in the field of regulatory biology. The project will also have strong educational impact, as it emphasizes data reproducibility and scientific ethics in three contexts: curricular enrichment for undergraduates who will integrate the research in their laboratory courses; annual outreach workshops for 7th grade minority students focused on science, technology, engineering and mathematics; and direct participation in research by early-career scientists at postdoctoral, graduate, and undergraduate levels. The mobility and mutagenic potential of transposable elements depends on mRNAs produced by synthesis of RNAs (transcription) from the elements themselves. This transcription step is a key control point for regulating transposable element mobility. Previous work has shown that transcription of transposable elements is turned off by small RNAs, which initiate silencing by a post-transcriptional interference mechanism that transitions to transcriptional-level silencing. This project will reproduce key aspects of that previous work and will apply modern technological capabilities to produce higher-value datasets that will be analyzed in creative and innovative ways to determine precisely how RNA interference is targeted to a particular transcript and how RNA-directed DNA methylation and transcriptional-level repression are established to produce trans-generational epigenetic silencing of transposable elements. Data and protocols will be broadly disseminated via open-access routes to enable the reproducibility of the research. The results should provide not only new understanding of how RNA-mediated silencing occurs, but also clues about how such silencing could be prevented. These insights could have practical consequences for genetically engineering plants with agronomic benefit.