Project Summary The overarching goal of this proposal is to develop a strategy to manipulate single mRNAs in live cell and use it to study post-transcriptional gene regulation, which is essential for cells to restrict proteins synthesis at the right time and place. It becomes a leading research focus because of its importance in learning, memory, development and other fundamental biological processes. Despite decades of research, the spatiotemporal dynamics of post-transcriptional regulation is poorly understood. This is due to the lack of experimental tools to control gene expression with high temporal resolution in subcellular compartments, such as leading edges of moving fibroblasts, anterior or posterior poles of developing oocytes, neuronal growth cones or dendritic spines. In this work, we propose to develop strategies to regulate gene expression at the single mRNA level in subcellular compartments. To achieve this goal, we will create optogenetic and chemigenetic tools to control single RNAs in live cells. First, we will use light- induced or chemical-induced dimerizer to tether protein factors onto target RNAs. Because proteins control the RNA metabolism, this allows us to regulate the fate of single mRNAs or modify the coded protein anywhere in a cell by precisely manipulating laser illumination or administering small molecules. Second, we will use chemically-modified light-sensitive guide RNA for the recently developed programmable RNA-targeting CRISPR-Cas13 technology. We plan to develop a light inducible RNA knock-down method and RNA binding proteins to modulate any endogenous RNA. We will use the technology to study the decay mechanism by synchronously induction of rapid RNA degradation. Combined with previously developed single mRNA translation assay in our lab, we will investigate the interplay between translation and decay machineries. By controlled RNA editing, we will visualize the distribution of newly synthesized proteins from single mRNAs in neuronal dendrites. Gene expression regulation plays a central role in all biological problems. The tool that the PI proposed here represents the ultimate spatiotemporal precision that one can manipulate when and where a gene is expressed. It is comparable to RNA interference technology, with added advantages of subcellular resolution, activating, repressive, and mRNA editing capability. The molecular biological reagents and microscopy tools will be applicable to a broad range of scientific community. This will allow us to address questions that cannot be answered before.

Narrative: We propose to develop optogenetic and chemigenetic tools to visualize and control the metabolism of single mRNAs with high spatiotemporal resolution in live cells. We will use the tools to investigate the molecular mechanism of RNA decay and localized translation. The experimental tools will be valuable for studying post-transcriptional gene expression which is important to decipher the molecular mechanism of neural development, learning and memory formation, and to develop therapeutic treatment for neurological diseases.