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

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.