Roles of Zpr1 in cellular proliferation and DNA/RNA metabolism

ZPR1 is a well conserved, essential protein in all eucaryotic organisms. ZPR1 was associated with various cellular functions, such as protein folding and translation regulation, transcription regulation, ribosome biogenesis and RNA transport and maturation. In the past years, ZPR1 was associated with neurodegenerative diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis type 4 (ALS4). ZPR1 was placed into the R-loop resolution complex as its interaction with Senataxin (SETX) is crucial for maintaining R-loop homeostasis in mammalian cells. However, the exact molecular mechanism of ZPR1 is still unknown. In this work we aim to investigate the essential function of Zpr1, to identify its role on chromatin while understanding its mechanism in regulating gene expression and DNA replication. To this end, we have used Saccharomyces cerevisiae as model system, taking advantage of the well conserved nature of Zpr1. To address the essential function of Zpr1, we have established a conditional depletion system, achieving fast and stringent depletion of the protein. Mapping the Zpr1 interactome we have found that Zpr1 associates with replisome components, such as Ctf4, Chl1 and Sen1. Examining the effect of Zpr1 loss on cellular proliferation we found that cells accumulate at the G1/S boundary and have shown that this arrest is not due to lethality caused by translational defects. Zpr1 is present on chromatin in all cell cycle stages, most abundantly in G1 and S phases. Total transcriptome and chromatin proteome analysis confirmed that Zpr1 loss in S phase leads to delayed cell cycle progression and suggested defects in various transcriptional processes. We discovered that Zpr1 depleted cells present severe replication initiation defects paired with delayed checkpoint activation. Strikingly, Zpr1 depletion did not result in defective resolution of transcription replication conflicts, while ChIP-seq analysis revealed significant overlap between Zpr1 and Sen1 chromatin locations. We suggest that Zpr1 has a significant role as a transcriptional regulator that may be linked with its roles in replication initiation. Loss of Zpr1 causes general decrease in transcription, decreasing R-loop levels and alters the expression of crucial factors, potentially linked to defective replication initiation.

Funder

Fondazione AIRC

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)