A mechanistic study of TRAIP mobilisation in UV response

Abstract

TRAIP is a primordial dwarfism-associated E3 ubiquitin ligase with a role in maintaining genome integrity. TRAIP localises in the nucleolus, but mobilises to stressed replication forks through its interaction with PCNA in response to UV. However, the underlying mechanism of this translocation remains unknown. In this study, I found that although PCNA-binding is required for TRAIP enrichment at UV-induced DNA damage foci, it is dispensable for TRAIP release from the nucleolus. I therefore postulated that TRAIP nucleolus-nucleoplasm translocation may be distinctly regulated. Examining the sub-cellular localisation of a panel of TRAIP mutants supported this idea. Moreover, by monitoring TRAIP-GFP dynamics in living cells, I also showed that TRAIP proteins dynamically shuttle between the nucleolus and nucleoplasm, prompting the identification of the determinants involved in sequestering TRAIP in the nucleolus. Strikingly, chemical inhibition of rDNA transcription resulted in TRAIP diffusion into the nucleoplasm, raising the possibility that rDNA transcription may facilitate TRAIP nucleolar retention. Accordingly, UV suppressed rDNA transcription and led to substantial decrease in nucleolar R-loops. Moreover, nucleic acid digestion using DNase I or RNase A released TRAIP from the nucleoli. These lines of evidence support a model in which TRAIP enrichment in the nucleolus requires active rRNA synthesis, and that TRAIP may associate with DNA/RNA hybrid structures during rDNA transcription. My study defines a bipartite mechanism that regulates TRAIP redistribution from the nucleolus to the nucleoplasm in response to UV damage, and highlights the nucleolus as a stress sensor in the DNA damage response that mediates protein trafficking.