Regulatory nucleic acid interactions and telomere complexes

This MIRA award request will unify two areas of R01-funded research in the laboratory of Deborah Wuttke at the University of Colorado Boulder. The common theme is probing the multi-layer regulatory networks that modulate gene expression and cellular homeostasis through regulation by nucleic acids. Specifically, the program will address outstanding questions regarding the non-canonical recognition of RNA by DNA-binding transcription factors (TFs) and the management of ssDNA by replication A (RPA) type factors. During the last grant period, we discovered a robust, and unexpected, RNA-binding activity inherent in numerous transcription factors. This RNA-binding activity was found to be mediated by extended versions of their DNA-binding domains and directly compete with DNA binding. Furthermore, we found that this activity was structure-specific rather than sequence specific in vitro. These observations raise critical questions regarding the role of RNA binding by TFs in transcriptional regulation and provide the direct motivation for the research program described here. The next steps are to further define and understand the specificity for the in vivo RNA targets and determine the impact TF RNA interactions has on the transcriptional program. We will take advantage of biochemically validated separation of function mutants that allow us to independently disrupt either RNA- or DNA-binding activity in cells and probe their mechanism of action using integrated transcriptomics and single molecule imaging. The impact of this program is high because understanding the roles of genome-wide pervasive transcription and how this activity influences diverse cellular processes remains a major unanswered question in biology. Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are required for chromosome stability and cellular proliferation. These structures are essential for human health because dysregulation of either telomere protection or telomerase activity causes many inherited and acquired human diseases, with telomere dysfunction closely tied to cancer and aging. Our program is focused on two protein complexes that manage ssDNA in many chromosomal contexts, including at telomeres and replication forks. Human CST is a heterotrimeric protein complex that protects and maintains sites of G-rich ssDNA throughout the genome, acting prominently at telomeres through binding the conserved G-rich overhang to coordinate the activities of telomerase and Pol-alpha primase. Mutants of CST are associated with a range of human diseases characteristic of proliferation defects. The structural, functional and biochemical parallels between the CST and RPA complexes suggest a highly tuned interplay of their activities that allow for their crosstalk in the management of difficult G-rich regions of chromatin and our recent data point to the intricate integration of the general DNA-maintenance and telomere machineries. This is key to understanding the basic biology of chromosome maintenance and the catastrophic consequences of its misregulation. Project Number: 1R35GM162595-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: DEBORAH WUTTKE | Institution: UNIVERSITY OF COLORADO, Boulder, CO | Award Amount: $495,480 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-J (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259345