Oral Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

Determination of the structure and binding substrates of ESRP1 reveals multiple modes of target regulation (143998)

Dawei Liu 1 , B. Kate Dredge 1 , Andrew G Bert 1 , Katherine A Pillman 1 , John Toubia 1 2 , Wenting Guo 3 , Boris J.A Dyakov 4 5 , Melodie M Migault 1 , Daniel P Neumann 6 , Saba Montazaribarforoushi 7 8 , Chun-Xia Zhao 9 , Lachlan A Jolly 7 10 , Yeesim Khew-Goodall 1 , Philip A Gregory 1 , Anne-Claude Gingras 4 5 , Dinshaw Patel 11 , Baixing Wu 3 , Gregory J Goodall 1
  1. Centre for Cancer Biology, an alliance of SA Pathology and Adelaide University , Adelaide, SA, Australia
  2. Data and Bioinformatics Innovation, Department of Genetics and Molecular Pathology, Adelaide, SA, Australia
  3. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, RNA Biomedical Institute, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China
  4. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University Ave, Toronto, Canada
  5. Department of Molecular Genetics, University of Toronto, Toronto, Canada
  6. School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia
  7. Adelaide Medical School, Adelaide University, Adelaide, SA, Australia
  8. Robinson Research Institute, Adelaide University, Adelaide, SA, Australia
  9. School of Chemical Engineering, Faculty of Science, Engineering and Technology, Adelaide University, Adelaide, SA, Australia
  10. School of Pharmacy and Biomedicine, Adelaide University, Adelaide , SA, Australia
  11. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Epithelial Splicing Regulatory Protein 1 (ESRP1) orchestrates epithelial-specific gene expression programs, yet the mechanisms underlying its dual nuclear–cytoplasmic functions remain unclear. Here, crystal structures of the ESRP1 RNase-H-like and qRRM domains reveal a conserved double-clamp mechanism for 5′-GGU-3′ motif recognition across all three qRRM domains and establish the RNase-H-like domain as catalytically inactive. Our first genome-wide HITS-CLIP map of ESRP1 in human cells confirms GGU recognition and shows that nuclear ESRP1(nESRP1) preferentially binds introns to regulate alternative splicing, while cytoplasmic ESRP1(cESRP1) predominantly targets 3′UTRs. cESRP1 constitutes 60–90% of the two major full-length isoforms across epithelial tissues, a pool that nESRP1 generates through an MBNL1/2-dependent autoregulatory splicing circuit. Functionally, both isoforms promote mRNA decay through 3′UTR binding, while nESRP1 additionally directs alternative splicing toward non-coding variants including NMD substrates and non-coding linear transcripts – a regulatory logic we term "alternative non-coding splicing" (ANS); systematic classification of 71 post-transcriptionally regulated targets identifies 18 high-confidence ANS targets and 8 cytoplasmic 3′UTR decay targets. Both activities converge on the core NMD factor UPF1 through direct interaction with ESRP1 and require the catalytically inactive RNase-H-like domain. These findings establish ESRP1 as a bifunctional regulator that coordinates epithelial gene expression through complementary splicing and UPF1-dependent mRNA decay, with a catalytically inactive RNase-H-like domain essential for both activities.