Understanding mechanisms of RNA polymerase II transcriptional termination

/ABSTRACT Proper regulation of mRNA transcription is essential for normal cellular metabolism, and mis-regulation can lead to multiple pathologies and disease states. In recent years it has become clear that transcriptional termination at the 3' ends of genes is mis-regulated in response to cellular stress and some diseases. For example, in response to a short heat shock, human RNA polymerase II (Pol II) reads through the typical 3' end of the vast majority of mRNA genes and continues transcribing for many kilobases into intergenic regions and neighboring genes. The mechanisms that control transcription termination in response to cellular stress are largely unknown. Importantly, transcriptional 3' end read-through also occurs at thousands of genes in healthy human tissues and cell types. The parameters that dictate whether a given gene exhibits read-through transcription during normal cellular metabolism are not clear. The proposed research will reveal mechanisms that control how read-through transcripts are generated in human cells both in response to heat shock, and in the non-stressed state. At its core, termination of Pol II transcription involves two steps – cleavage of the nascent RNA to generate the 3' end of the mRNA and removal of the polymerase from the genome – both of which will be studied. In Specific Aim 1 experiments will uncover mechanisms that regulate the RNA cleavage step of transcription termination in response to heat shock. Supporting data show that after heat shock the cleavage step of termination is defective at the majority of genes; however, over a thousand genes show proper cleavage during heat shock despite having extensive read-through. The proposed research will reveal the mechanisms by which 3' end mRNA cleavage is differentially controlled in a gene-specific fashion in response to heat shock. Experiments in Specific Aim 2 will determine mechanisms that regulate Pol II displacement from the genome after RNA cleavage. Preliminary experiments have identified genes in non-heat shocked and heat shocked cells where cleavage and read- through are uncoupled. These genes exhibit strong RNA cleavage, but also show extensive read-through transcription, suggesting termination at these genes is regulated via removal of Pol II from the genome. These gene sets will be used to understand what controls the displacement of Pol II after proper cleavage, with a focus on the movement of the polymerase and the activity of the exonuclease Xrn2, leveraging a combination of cell-based experiments and in vitro single molecule studies. Together the proposed studies define mechanisms controlling transcriptional termination in cells growing normally and in response to cellular stress. Project Number: 1R01GM158787-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: James Goodrich | Institution: UNIVERSITY OF COLORADO, Boulder, CO | Award Amount: $307,854 | Activity Code: R01 | Study Section: Molecular Genetics Study Section[MG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11296164