Fluorescent reporters for real time single cell Pol III transcription measurement

SUMMARY RNA polymerase III Pol III is increasingly recognized as an important drug target and a rapidly developing area of academic research Pol III is responsible for generating RNAs that are critical for cell growth such as tRNAs certain rRNAs and various noncoding RNAs involved in processes such as mRNA splicing These RNAs are required for cell growth proliferation and differentiation and disrupting the pathways that control Pol III is now thought to be valuable for targeting cancer and obesity Despite the importance of Pol III little is known about the pathways that regulate Pol III transcription This is largely due to the absence of simple and robust reporters for Pol III activity This is in contrast to reporter assays for Pol II promoters for mRNA transcription these reporters comprise Pol II promoters controlling reporter genes such as green fluorescent protein and have been the major driver for drug discovery for mRNA transcription inhibitors However Pol III generates RNAs that cannot be translated As a result nearly all studies of Pol III rely on Northern blotting which is slow and not suited to high throughput screening efforts At Lucerna we have developed a photostable RNA mimic of red fluorescent protein termed Corn and demonstrated that this RNA can serve as a robust fluorescent reporter for Pol III promoters By simply creating a Pol III promoter that controls the synthesis of the Corn RNA fluorescence can be readily detected without the need to synthesize a protein We have demonstrated this concept using the S subtype of a Pol III promoter Interestingly we found marked cell to cell variation in Pol III transcription after cells are exposed to serum and other growth promoting signals Therefore the goal of this Phase I SBIR project is to develop this simple and genetically encodable Corn reporters for live cell imaging and measurement of all three subtypes of Pol III transcription Further we will develop this reporter system so that it can be used in flow cytometry analysis allowing Pol III transcription to be monitored in hundreds of thousands of cells over time For phase II we will adapt our Pol III reporters into marketable toolkits validate the Pol III reporters in disease relevant cell lines and use the Cas CRISPR technology to image endogenous Pol III transcription activity Together the plasmids and kits generated in this project will result in the first Pol III reporters and will therefore constitute an important commercial product that will have wide utility in the academic RNA research community and will enable discovery of small molecule inhibitors of this previously hard to assay class of drug targets PROJECT NARRATIVE RNA polymerase III Pol III is responsible for of total cellular transcription and Pol III activity generates critical RNAs that have been linked to cell homeostasis and disease pathogenesis The lack of real time imaging of Pol III transcription dynamics in cells has limited our ability to understand Pol III activation and the cell to cell variability in Pol III gene expression that occurs in response to stimuli In this project Lucerna will develop a simple genetically encodable fluorescence reporter for monitoring Pol III transcription in real time in live cells which will provide a valuable research tool to study Pol III dynamics and to enable drug discovery targeting Pol III transcription pathways