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

合作研究：创意实验室：用于表观遗传信号放大的非编码 RNA 的合理设计

• 批准号: 2243665
• 负责人: Karmella Haynes
• 金额: $ 211.05万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2243665&HistoricalAwards=false
• 关键词: Collaborative; Research; Ideas; Lab; Rational

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.

已经取得了相当大的进步来照亮基因组的暗物质 - 那些不编码蛋白质的段。这些非编码区域中的一些被转录为产生长的非编码RNA（LNCRNA），这些RNA与蛋白质编码的信使RNA相似。尽管通常仅在细胞内发现它们，但在无数的生物过程中隐含了LNCRNA，包括细胞生长和生存，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固有的生物技术。该项目将使用合理的“理解”方法来合理设计，并使用lncrnas控制大量的染色体DNA。通过在人类细胞和酵母中使用这种新的RNA技术来证明广泛的效用（真菌）。控制其活动的工程LNCRNA不仅有助于对这些神秘的RNA的基本理解，而且还会利用巨大的潜力，以在跨物种的农业和医疗应用中产生有用的生物分子。该项目包括独特的量身定制的编程，以支持从高中生到博士后研究员的参与者的STEM劳动力开发。 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.最终目标是对LNCRNA可以以染色体尺度执行沉默效应的机制获得基本理解，尽管在细胞中以低水平表达。为了解决这个问题，研究人员将首先测试不同重复序列对哺乳动物细胞培养系统沉默能力的影响。然后，他们将尝试通过将合成的LNCRNA集成到其他染色体或通过基于CRISPR的靶向靶向来沉默常染色体基因座基因座。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能够使用酵母遗传学的力量进一步测试和优化沉默系统。最后，该项目将使用数据来指导合成沉默系统的合理设计，这些系统可以容易部署在哺乳动物细胞中，以实现靶向和可调的沉默。总体而言，研究人员期望突破核中LNCRNA功能的当前理解的边界，预期的结果包括定义稳定和特定表观遗传调节所需的最小成分和最小的LNCRNA水平。通过从基础上重构功能组件，他们希望验证，挑战和/或扩大涉及RNA的基本力学。鉴于可调节的LNCRNA调制可能是广泛适用的生物技术，因此这项工作对生物工程具有令人兴奋的影响。该协作项目是由NSF赞助的想法实验室造成的。它将由新兴领域计划和分子和细胞生物科学的部门共同资助。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，通过评估来诚实地支持支持。