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: 2243666
• 负责人: Keriayn Smith
• 金额: $ 66.58万
• 依托单位: University of North Carolina at Chapel Hill
• 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=2243666&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.

在阐明基因组的暗物质方面已经取得了相当大的进展——那些不编码蛋白质的片段被转录产生长非编码RNA（lncRNA），它们的加工过程与编码蛋白质的信使RNA类似。尽管lncRNA在细胞内的含量通常很少，但它与多种生物过程有关，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。该项目将利用这些 lncRNA 潜在固有的生物技术，通过使用这种新的 RNA 技术来合理设计和使用 lncRNA 来控制大片染色体 DNA，从而证明其广泛的实用性。对人类细胞和酵母（真菌）中的 lncRNA 进行工程改造以控制其活性不仅有助于对这些神秘 RNA 的基本了解，而且还可以挖掘在农业和医学领域产生有用生物分子的巨大潜力。该项目包括专门定制的计划，以支持从高中生到博士后的参与者的 STEM 劳动力发展，并将重点关注 RNA 生物学的新虚拟和实践活动与成功的 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，以充分表征的 Xic 位点及其相关的 lncRNA Xist 为基础，Xist 负责沉默女性中的一条 X 染色体最终目标是从根本上了解 lncRNA 在染色体水平上发挥沉默作用的机制。为了解决这个问题，研究人员将首先测试不同的影响。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位点。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，以便进一步测试和优化沉默系统。最后，该项目将利用这些数据来指导合成沉默系统的合理设计，该系统可以轻松地部署在哺乳动物细胞中，以实现靶向和可调节的沉默。总体而言，研究人员希望突破沉默的界限。目前对细胞核中 lncRNA 功能的理解，预期结果包括定义稳定和特异性表观遗传调控所需的最小成分和 lncRNA 水平，他们希望通过从头开始重建功能成分来验证、挑战和/或。鉴于可调节的 lncRNA 调节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影响。该合作项目由 NSF 赞助的创意实验室共同资助。新兴前沿计划和分子与细胞生物科学部。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。