Defining New Roles of the E3 Ligase RNF25 in DNA Replication and Stress Signaling

About This Grant

ABSTRACT All growing cells experience DNA Replication Stress (RS, a slowing of DNA synthesis), which poses a major threat to genome integrity and viability. To safeguard against RS, stalled replication forks trigger DNA Damage Response (DDR) signaling cascades which coordinate resolution of stalled forks with cell cycle progression and other processes to ensure cellular homeostasis and survival. DDR defects can lead to severe human health consequences including stem cell dysfunction, aging, neurodegeneration, neoplasia, immune deficiencies, and developmental syndromes. While DDR signaling is clearly crucial for recovery from RS, the ways in which the many effector branches of the DDR are activated, coordinated with each other, and integrated with other cellular processes to orchestrate cell fate decisions are poorly understood. In exciting recent work we identified the RING finger E3 ligase RNF25 as a major new replication fork-localized DDR factor that is critically required for replicating cells to tolerate RS. Previous studies showed that RNF25 ubiquitylates ribosomal proteins to alleviate ribotoxicity (RNA damage). Remarkably, we demonstrated that RNF25 remediates RS independently of its canonical role in regulating ribosome function. Thus RNF25 is the first example of a `dual stress responder' that mediates responses to both genotoxicity and ribotoxicity. Our discovery of RNF25 as a major new DDR factor reveals large gaps in our knowledge of genome maintenance. The objective of this application is to define how RNF25 is regulated, and how it interfaces with other components of the DDR network and the ribotoxic stress response to alleviate RS. Our separation-of-function RNF25 mutant also provides an outstanding opportunity to determine the relative contribution of genotoxicity vs. ribotoxicity to cell fate decisions. The rationale is that we will mechanistically define a major new effector branch of the DDR (mediated by RNF25), and we will reveal how it is coordinated with other pathways to ensure genome integrity and survival. Our central hypothesis is that RNF25 critically interacts and cooperates with several important DNA repair proteins (REV7, PARPs, and MAGE-A4) to resolve RS. Our Specific Aims (SAs) are: SA1 Define how RNF25 associates with REV7 to orchestrate responses to DNA-damaging agents. SA2 Establish PARPs as mediators of RNF25 signaling in the DDR. SA3 Establish the E3 ligase cofactor MAGE-A4 as a regulator of RNF25 signaling. We will use biochemical approaches to define mechanisms by which RNF25 associates with its partner proteins. We will use unbiased screens to define genetic interactions between RNF25 and other major DDR pathways. We will use phenotypic endpoints (such as DNA synthesis at single DNA fiber resolution, cell cycle progression and viability) to define how RNF25 and its interacting proteins and pathways dictate cell fate. We propose innovative solutions to important problems such as `How do cells activate the DDR?' and `How are different branches of the DDR integrated and coordinated?'. The proposed work is significant because we will mechanistically define an important new branch of the DDR.