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In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells
Published on: May 1, 2019
Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding
Panagiota Kafasla1, Nina Morgner, Carol V Robinson
1Department of Biochemistry, University of Cambridge, Cambridge, UK.
The EMBO Journal
|September 23, 2010
Summary
Polypyrimidine tract-binding protein (PTB) binding to the poliovirus internal ribosome entry site (IRES) was studied. PTB binding enhances IRES activity by positioning eukaryotic initiation factor 4G (eIF4G) for optimal ribosome entry.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The poliovirus internal ribosome entry site (IRES) directs cap-independent translation initiation.
- Polypyrimidine tract-binding protein (PTB) is known to regulate IRES activity, but its precise binding orientation and mechanism remain unclear.
- Understanding PTB-IRES interactions is crucial for deciphering viral translation control.
Purpose of the Study:
- To determine the binding orientation of PTB to the PV-1(M) IRES.
- To identify which RNA-binding domains (RBDs) of PTB interact with specific IRES regions.
- To elucidate the interplay between PTB and eukaryotic initiation factor 4G (eIF4G) during IRES-mediated translation.
Main Methods:
- Tethered hydroxyl-radical probing was employed to map PTB binding sites on the PV-1(M) IRES.
- Mass spectrometry was used to confirm PTB-IRES complex stoichiometry.
- Investigated the influence of eIF4G on PTB binding and vice versa.
Main Results:
- A single PTB molecule binds to the PV-1(M) IRES under conditions of strong IRES stimulation.
- PTB RNA-binding domains 1 and 2 (RBDs 1 and 2) interact with the Domain V stem-loop.
- PTB RBDs 3 and 4 interact with single-stranded regions adjacent to Domain V, near the eIF4G binding site.
Conclusions:
- PTB binding to the PV-1(M) IRES is specific and involves multiple RBDs.
- PTB binding is subtly modulated by eIF4G, and eIF4G binding is affected by PTB.
- PTB likely enhances PV-1(M) IRES activity by facilitating optimal eIF4G positioning for ribosome recruitment.
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