Investigating the impact of PAD4-mediated citrullination on the E2F genomic landscape

Thesis

E2F1 is a master transcription factor that plays a pivotal role in regulating cell cycle progression and proliferation, as well as mediating other cell fates including apoptosis and the inflammatory response. These diverse biological outcomes of E2F1 activity are determined in part by post-translational modifications, including arginine methylation mediated by protein arginine methyltransferases (PRMT) 1 and 5, and citrullination mediated by peptidyl arginine deiminase (PAD) 4. The activity of PRMT5 and PRMT1 are antagonistic to one another; the former symmetrically methylates R111/R113 of E2F1 and favours proliferation, whilst the latter promotes E2F1-dependent apoptosis via asymmetric methylation at R109. On the other hand, PAD4 channels E2F1 into a pro-inflammatory pathway by citrullinating its target arginine residues, which also include R109. Recently, PRMT5-driven methylation events at R111/R113 and recruitment of the reader protein p100/TSN were found to permit E2F1 to regulate many genes at the level of alternative splicing. However, despite mounting evidence suggesting that many RNA-binding proteins are substrates for citrullination, the link between PADs and RNA processing remains unclear. In this study, by performing RNA sequencing, we demonstrated that E2F1 knockdown and PAD4 inhibition exhibit significant global impacts on the human cancer transcriptome at the level of alternative RNA splicing. Moreover, we uncovered that this regulation involves the citrullination of E2F1, which enhances its interaction with p100/TSN and the splicing factor SRSF3. Using RNA immunoprecipitation methods, we additionally elucidated that PAD4 plays an essential role in regulating the RNA binding affinity of SRSF3 in an E2F1-dependent manner, which underpins the molecular mechanism by which PAD4 and E2F1 together regulate alternative splicing. In summary, we discover a new function of PAD4 as a splicing regulator in cancer cells and propose a mechanism by which this alternative splicing is mediated by the novel interplay between E2F1 and SRSF3.

Authors: Harada, K

• DOI: 10.5287/ora-xobx2o6aj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: post-translational modifications; E2F1; RNA splicing; cancer; citrullination
• Subjects: Molecular biology; Oncology