RUI: Early Endings - Characterizing the Role of Hrp1 in RNA Polymerase II Transcription Attenuation

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Life depends on information stored in DNA, which is expressed into RNA and proteins that perform cellular functions. An initial step of gene expression is transcription, where a molecular machine called RNA polymerase reads DNA to synthesize RNA. As RNA polymerase moves along DNA, it can be interrupted by regulatory stop signals that terminate transcription prematurely. Downregulation of gene expression by early transcription stoppage occurs widely in cells ranging from bacteria to human, but the underlying mechanism and selectivity remains unclear. This research project will investigate premature transcription termination in the yeast S. cerevisiae, a tractable model for studying many conserved biological processes. Undergraduate student researchers will be trained to use classical genetics and modern bioinformatic tools to dissect transcription stop signals and discover mutants that alter recognition. New gene targets will be identified, with a broader goal of identifying shared regulatory features. Student trainees will have opportunities to present their work at national research conferences and coauthor publications. This research will also be incorporated into a core undergraduate biology laboratory course, mobilizing 50 additional students to validate gene targets. Course resources will be shared broadly so additional educational communities may contribute and benefit.Premature transcription termination (attenuation) of eukaryotic RNA Polymerase II (Pol II) is more prevalent than once appreciated but remains ill-defined. The investigators hypothesize that a hybrid transcription termination pathway involving the Hrp1 RNA-binding protein and Sen1 helicase contributes broadly to yeast Pol II attenuation. This research project will generate a comprehensive genetic profile of ten newly identified attenuators and identify the larger repertoire of Hrp1-dependent attenuators across the yeast genome. In Aim 1, a genetic selection will identify cis-acting mutations that disrupt attenuator function in plasmid-based reporter genes, followed by confirmation in CRISPR-edited genomic DNA. A genetic screen will probe the effect of trans-acting mutations that alter termination factor recruitment, Pol II CTD modification, and Pol II pausing. A direct mechanism will be assayed using Hrp1 mutants defective for binding RNA or affiliated proteins. In Aim 2, an auxin-inducible degron system will deplete Hrp1, followed by precision nuclear run-on sequencing (PRO-Seq) to monitor genome-wide Pol II read-through defects. This work will inform analysis of the Hrp1 ortholog HNRNPDL, a human protein that likewise binds AU-rich RNA and regulates transcription. In addition, engineered attenuators may be harnessed for dynamic gene control in biotechnology applications, including yeast expression of industrial enzymes.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.