The Role of Upf1 in Telomere Homeostasis

Abstract

Telomeres are specialized DNA structures at the ends of chromosomes that protect against the loss of genetic information during replication. Although the protein Upf1, involved in both mRNA surveillance and genomic stability, has been implicated in telomere maintenance, its exact role remains unclear. This study aimed to examine the roles of Upf1 at telomeres and in genomic stability using an inducible HeLa cell line expression system (FLP-IN). After inserting fluorescent tags like the Kaede tag to track UPF1 in live HeLa T-REx cells, we visualized UPF1 dynamics and studied its functions more effectively. The research began with the validation of starting materials, including Kaede-N1 and pcDNA5-FRT-TO-CAT-FLAG-UPF1 plasmids, confirming their identity. The hygromycin sensitivity test validated the HeLa T-REx cell line, proving its suitability for the Flip-In system. The characterization of HeLa T-REx cell growth dynamics was examined, and optimal seeding densities for transfection were optimized. The average doubling time for HeLa T-REx cells was approximately 24 hours. To generate an inducible HeLa cell line expression system (FLP-IN), the project created novel cell lines expressing fluorescently tagged UPF1. A pCDNA-FRT-TO-CAT-FLAG- UPF1-Kaede construct was created through a two-step cloning process. First, UPF1 was cloned into Kaede-N1, creating an ~8.1 Kb Kaede-N1-UPF1 construct. Then, Kaede- UPF1 was cloned into pcDNA-FRT-TO-CAT, resulting in the final ~10 Kb construct. The final construct, pCDNA-FRT-TO-CAT-FLAG-UPF1-Kaede, was confirmed with restriction mapping and sequencing. Although site-directed mutagenesis to remove two stop codons was unsuccessful, hygromycin selection indicated successful construct integration. Despite high transformation efficiency for the control plasmid, no colonies were obtained from the DpnI-treated DNA samples, suggesting issues with the mutagenesis reaction itself. The study proceeded to create a stable FLP-IN cell line expressing fluorescently tagged UPF1 in HeLa T-REx cells. Colonies appeared under hygromycin selection, indicating successful integration of the construct. However, the presence of unremoved stop codons may hinder the production of the desired UPF1-Kaede fusion protein. An important accomplishment was the effective establishment of a doxycycline-inducible method for managing the expression of FLAG-tagged UPF1. The Western blot analysis showed induction in the expression of FLAG-tagged UPF1 protein at around 130 kDa in samples treated with doxycycline at 24, 48, and 72 hours after induction. Endogenous UPF1 was seen in both induced and non-induced samples, indicating that the presence of doxycycline did not impact endogenous UPF1. This suggests the possibility of investigating the impact of UPF1 overexpression on cellular functioning. This study provides valuable tools and insights for investigating Upf1's role in telomere maintenance and genomic stability. The inducible expression method enables precise regulation of Upf1 levels, allowing future investigations into its interactions at telomeres. Although faced with difficulties in site-directed mutagenesis and the creation of stable cell lines that produce the complete UPF1-Kaede fusion protein, the experiment has effectively built the basis for future investigations into the involvement of Upf1 in telomere biology and its possible significance in cancer research. Future research should prioritize improving mutagenesis techniques and investigating UPF1's interactions at telomeres to further our comprehension of genomic integrity preservation.