Collaborative Research: Ideas Lab: Rational Design of Noncoding RNA for Epigenetic Signal Amplification

在阐明基因组的暗物质方面已经取得了相当大的进展-这些片段不编码蛋白质。这些非编码区中的一些被转录以产生长的非编码RNA（lncRNA），其加工类似于蛋白质编码信使RNA。尽管lncRNA通常仅在细胞内少量存在，但它们涉及无数生物过程，包括细胞生长和存活，细胞身份和环境相互作用，以及许多人类和动物疾病。下一个前沿将是利用这些lncRNA潜在固有的生物技术。该项目将使用一种合成的“通过构建来理解”的方法来合理设计和使用lncRNA来控制大片染色体DNA。通过在人类细胞和酵母（真菌）中使用这种新的RNA技术，将证明其广泛的实用性。工程化lncRNA以控制其活性不仅有助于对这些神秘RNA的基本理解，而且还可以挖掘在跨物种的农业和医学应用中产生有用生物分子的巨大潜力。该项目包括独特的定制编程，以支持从高中生到博士后研究员的参与者的STEM劳动力发展。新的虚拟和实践活动将专注于RNA生物学，并与成功的STEM推广计划相结合，以确保产生强大的影响。该项目旨在定义由lncRNA介导的大规模沉默所需的最小特征，建立在已充分表征的Xic基因座及其相关lncRNA Xist的基础上，Xist负责沉默雌性哺乳动物和有袋动物的X染色体之一。最终目标是获得对lncRNA在染色体规模上发挥沉默作用的机制的基本理解，尽管lncRNA在细胞中以低水平表达。为了解决这个问题，研究人员将首先在哺乳动物细胞培养系统中测试不同重复序列对沉默能力的影响。然后，他们将尝试通过将合成的lncRNA整合到其他染色体中，或通过基于CRISPR的靶向来沉默常染色体基因座。我们将使用这些数据来参数化数学模型，以更好地了解沉默过程的动态和局限性。他们还将使用Xist系统的组件在单倍体酵母细胞中创建一个独立的沉默系统。这将使详细的遗传操作，以进一步测试和优化沉默系统使用酵母遗传学的力量。最后，该项目将使用这些数据来指导合成沉默系统的合理设计，这些系统可以很容易地部署在哺乳动物细胞中，以实现靶向和可调沉默。总的来说，研究人员希望推动目前对细胞核中lncRNA功能的理解的界限，预期的结果包括定义稳定和特异性表观遗传调控所需的最小组分和lncRNA水平。通过从头开始重建功能组件，他们希望验证，挑战和/或扩展涉及RNA的基本机制。考虑到可调lncRNA调制可能是一种广泛适用的生物技术，这项工作对生物工程具有令人兴奋的意义。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Considerable progress has been made to illuminate the dark matter of the genome - those segments that do not encode for protein. Some of these noncoding regions are transcribed to produce long noncoding RNAs (lncRNAs) that are processed similarly to protein-encoding messenger RNAs. Although they are often found only at low amounts within a cell, lncRNAs have been implicated in a myriad of biological processes including cell growth and survival, cell identity and environmental interactions, as well as numerous human and animal diseases. The next frontier will be to tap into the biotechnology potentially inherent to these lncRNAs. This project will use a synthetic, ‘understanding by building’ approach to rationally design and use lncRNAs to control large swaths of chromosomal DNA. Broad utility will be demonstrated by using this new RNA technology in human cells and in yeast (fungi). Engineering lncRNAs to control their activity will not only contribute to fundamental understanding of these enigmatic RNAs, but also tap the vast potential to generate useful biomolecules in agricultural and medical applications across species. The project includes uniquely tailored programming to support STEM workforce development for participants ranging from high school students to postdoctoral fellows. New virtual and hands-on activities focused on RNA biology will be integrated with successful STEM outreach programs to ensure strong impact.This project seeks to define the minimal features that are required for large-scale silencing mediated by lncRNA, building off of the well-characterized Xic locus and its associated lncRNA, Xist, which is responsible for silencing one of the X chromosomes in female mammals and marsupials. The ultimate goal is to gain fundamental understanding of the mechanisms by which lncRNAs can exert silencing effects at a chromosomal scale despite being expressed at a low level in the cell. To tackle this, the investigators will first test the effects of different repeats on silencing ability in a mammalian cell culture system. They will then attempt to silence autosomal loci by integrating synthetic lncRNAs into other chromosomes, or via CRISPR-based targeting. We will use these data to parameterize mathematical models to better understand the dynamics and limitations of the silencing process. They will also create a stand-alone silencing system in haploid yeast cells using components of the Xist system. This will enable detailed genetic manipulations to further test and optimize the silencing system using the power of yeast genetics. Finally, the project will use the data to guide rational design of synthetic silencing systems that can be readily deployed in mammalian cells to achieve targeted and tunable silencing. Overall, the investigators expect to push the boundaries of the current understanding of lncRNA functions in the nucleus, with anticipated outcomes to include defining the minimal components and lncRNA levels required for stable and specific epigenetic regulation. By reconstituting functional components from the ground-up, they expect to validate, challenge, and/or expand fundamental mechanics involving RNAs. Given that tunable lncRNA modulation could be a broadly applicable biotechnology, this work has exciting implications for bioengineering.This collaborative project resulted from an NSF-sponsored Ideas Lab. It will be co-funded by the Emerging Frontiers Program and the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.