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，可以通过插入新位置来动员和产生突变。为了防止这种诱变结果，细胞进化出了干扰转座元件动员的机制。先前的工作确定了小 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，因此该结果预计将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，因为它在三个方面强调数据可重复性和科学伦理：丰富本科生的课程，让他们将研究融入实验室课程；为七年级少数族裔学生举办年度外展讲习班，重点关注科学、技术、工程和数学；早期职业科学家在博士后、研究生和本科阶段直接参与研究。 转座元件的移动性和诱变潜力取决于元件本身合成 RNA（转录）产生的 mRNA。该转录步骤是调节转座元件迁移性的关键控制点。先前的研究表明，转座元件的转录被小RNA关闭，小RNA通过转录后干扰机制启动沉默，并转变为转录水平沉默。该项目将重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，以精确确定RNA干扰如何针对特定转录本，以及如何建立RNA指导的DNA甲基化和转录水平抑制以产生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途径广泛传播，以实现研究的可重复性。这些结果不仅可以提供对 RNA 介导的沉默如何发生的新理解，还可以提供有关如何预防这种沉默的线索。这些见解可能会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.