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

转座元件是基因组中的DNA片段，可以通过插入新的位置来动员和创造突变。为了防止这种突变结果，细胞已经进化出干扰转座元件动员的机制。以前的工作确定了小RNA在转座元件抑制中的作用。该项目将在模式植物拟南芥中使用现代基因组学方法，对导致普遍机械论观点的假设和结论进行“严格测试”，并扩大对转座元件沉默是如何触发的理解。由于转座元件广泛存在于植物和动物的基因组中，这一结果有望在调控生物学领域产生广泛的影响。该项目还将产生强大的教育影响，因为它在三个背景下强调数据的可重复性和科学道德：为将研究纳入其实验室课程的本科生提供丰富的课程；为7年级少数民族学生举办以科学、技术、工程和数学为重点的年度外联讲习班；以及直接参与博士后、研究生和本科生等早期职业科学家的研究。转座元件的迁移率和诱变潜力取决于从元件本身合成RNA(转录)所产生的mRNAs。这一转录步骤是调节转座元件迁移率的关键控制点。以前的工作表明，转座元件的转录是由小RNA关闭的，小RNA通过转录后干扰机制启动沉默，然后过渡到转录水平的沉默。该项目将再现先前工作的关键方面，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，以准确确定RNA干扰如何针对特定转录本，以及如何建立RNA指导的DNA甲基化和转录水平的抑制以产生可转座元件的跨代表观遗传沉默。数据和协议将通过开放获取的途径广泛传播，以使研究具有可重复性。这些结果不仅应该为RNA介导的沉默是如何发生的提供新的理解，而且还应该为如何防止这种沉默提供线索。这些见解可能会对具有农学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.