Characterizing quality control mechanisms required for nuclear export and cellular homeostasis - cellular health depends on gene expression patterns that fluctuate during development and in response…

Characterizing quality control mechanisms required for nuclear export and cellular homeostasis - cellular health depends on gene expression patterns that fluctuate during development and in response to environmental stressors, such as heat and nutrient starvation, through highly regulated RNA biogenesis pathways. During maturation, mrnas undergo several processes that involve the CO-transcriptional recruitment of RNA-binding proteins (RBPS). These processes include 5' capping, intron splicing, 3' end processing, and polyadenylation, ultimately leading to the packaging of the mrna into an export-competent messenger ribonucleoprotein complex (MRNP). Alterations in gene expression patterns associated with stress arise in part due to regulated and/or defective nuclear maturation of mrnas. Hallmarks of these disruptions include altered localization of nuclear components and/or nuclear retention of mrnas. Although the steps involved in normal mrna biogenesis have been individually well-characterized, how cells dynamically and systemically adapt to stress to reinstate nuclear homeostatic processes is not well understood. This GAP in knowledge necessitates the identification and characterization of processes and proteins that are required for the maintenance and restoration of nuclear export and gene expression following perturbation. In unpublished work as part of my PHD, I identified several proteasome subunits and pseudouridine synthases significantly enriched with poly(a)-rnas retained in the nucleus. Based on this observation, and the function of these components, I hypothesize that the proteasome and pseudouridine synthases function together to maintain nuclear homeostasis: the proteasome degrades aggregates of RBPS while pseudouridine synthases modify mrnas for storage and future export. These core quality control measures would be responsible for ensuring the maintenance of gene expression while integrating environmental cues to adjust gene expression programs accordingly. Additionally, in cases of RBP dysfunction, these mechanisms would help maintain nuclear homeostasis by regulating RNA levels and proteotoxicity. To test my hypothesis, I plan to use an innovative RNA-centric proximity labeling method in export mutants to enrich for components that colocalize with the nuclear poly(a)-RNA mass. This approach will help identify proteasome targets and characterize the degradation dynamics in the presence or absence of a functional nuclear proteasome. Additionally, I will combine this method with pseudo-SEQ to quantify pseudouridine modifications in rnas associated with the poly(a)-RNA mass in the presence or absence of the pseudouridine synthase enzyme, PUS7. Completing these aims will provide mechanistic insights into how cells overcome disruptions in nuclear mrna processing and export, which are essential for gene expression. Additionally, IT will shed light on gene-specific variations in nuclear mrna maturation, including quality control and surveillance mechanisms that lead to alternative processing or degradation. Ultimately, this work will lay the foundation for future research focused on understanding how disease-linked mutations can disrupt gene expression, contributing to the onset and progression of disease.