Role of Actinomycin D on the activity of RNA binding protein HuR

Date

2025-8-29

Author

Torun, Aydan

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Actinomycin D (Act D) is a polypeptide antibiotic that can bind to DNA and inhibit the elongation step of RNA polymerases. It is widely used as a transcription inhibitor in research and as a chemotherapeutic drug in clinical practice. Our preliminary data showed that short-term Act D treatment of HCT-116 and HeLa cell lines could increase the cytosolic translocation of the stabilizing RNA binding protein (RBP) HuR. We have used the Turbo-ID assay followed by LC-MS/MS and have identified a number of proteins known to be involved in RNA regulation in close proximity to HuR upon treatment with Act D (in HCT-116 cells). We have validated the interaction between HuR and the RBP FUS; the latter has been reported to play important roles in the formation and function of biomolecular condensates and aggregates. Immunocytochemistry analysis revealed that HuR can form punctate structures in the cytoplasm with Act D treatment. Moreover, the elevated cytoplasmic levels of HuR and FUS with Act D treatment decreased only when a hypotonic shock was applied, which is known to dissolve phase-separated proteins. Our findings suggest that Act D treatment and the resultant global cessation of transcription can activate a stress response in which HuR accompanied by FUS, translocate to the cytosol and interact with each other or other proteins to form RBP- enriched cytosolic condensates.

Subject Keywords

Act D, HuR, FUS, RBPs, Condensates

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https://hdl.handle.net/11511/116073

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Graduate School of Natural and Applied Sciences, Thesis