FRET-detectable interactions between the ARE binding proteins, HuR and p37AUF1
Pamela S David1, Rasheeda Tanveer, J David Port
1Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
RNA (New York, N.Y.)
|July 14, 2007
Summary
The study shows that mRNA-binding proteins HuR (Human antigen R) and AUF1 (AU-rich element binding factor 1) interact in the nucleus and cytoplasm, influencing gene regulation. Their interactions are dynamic, changing with cellular stress responses.
Area of Science:
- Molecular Biology
- Gene Regulation
- Post-transcriptional Modification
Background:
- Gene expression is tightly regulated post-transcriptionally, particularly at the mRNA stability level.
- A-U-rich elements (AREs) in 3' UTRs are key regulatory sites on mRNAs.
- HuR (Human antigen R) and AUF1 (AU-rich element binding factor 1) are critical ARE-binding proteins involved in mRNA stabilization and destabilization, respectively.
Purpose of the Study:
- To investigate the protein-protein interactions between HuR and AUF1.
- To determine if HuR and AUF1 can interact in both homodimeric and heterodimeric forms.
- To examine the cellular localization and dynamics of HuR/AUF1 interactions under various conditions.
Main Methods:
- Utilized cell biological and biophysical approaches, including fluorescence resonance energy transfer (FRET).
- Employed immunocytochemistry in live and fixed cells with fluorescently labeled CFP/YFP fusion proteins of HuR and p37AUF1.
- Monitored protein-protein interactions and cellular localization changes upon treatment with anisomycin and arsenite.
Main Results:
- Observed strong nuclear FRET signals, indicating homodimerization (HuR/HuR, AUF1/AUF1) and heterodimerization (HuR/AUF1).
- Anisomycin treatment induced rapid nuclear-to-cytoplasmic shuttling of HuR and slower shuttling of AUF1, with interactions detected in the cytoplasm post-shuttling.
- Arsenite treatment led to stress granule formation containing HuR and TIA-1, but notably excluded AUF1.
Conclusions:
- HuR and AUF1 proteins colocalize and functionally interact in both the nucleus and cytoplasm.
- The dynamic interactions and localization of HuR and AUF1 are influenced by cellular stimuli.
- FRET detection of AUF1/HuR interaction provides a novel method for studying mRNA-binding protein dynamics in RNA turnover.
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