A trypanosomatid protein specifically interacts with a mammalian iron-responsive element

Area of Science:

• Molecular Biology
• Evolutionary Biology
• Parasitology

Background:

• Iron homeostasis is crucial for cellular function in all eukaryotes.
• Iron regulation involves iron-regulatory proteins (IRPs) interacting with iron-responsive elements (IREs) on mRNA.
• Understanding the evolutionary origins of these regulatory mechanisms is key.

Purpose of the Study:

• To investigate the presence and characteristics of IRE-binding proteins in the ancient eukaryote Leishmania tarentolae.
• To compare the binding properties of the Leishmania protein with mammalian IRPs.
• To explore the evolutionary implications of IRE-RNA interactions.

Main Methods:

• Preparation of protein extracts from Leishmania tarentolae.
• Electrophoretic mobility shift assays (EMSAs) to detect protein-RNA interactions.
• Site-directed mutagenesis of mammalian IRE sequences.
• Determination of dissociation constants (Kd) for protein-IRE interactions.

Main Results:

• A Leishmania tarentolae protein specifically binds to mammalian IRE RNA.
• Mutations in the IRE that disrupt mammalian IRP binding also affect binding by the Leishmania protein.
• The dissociation constant for the Leishmania protein-IRE interaction (0.7±0.3 µM) is weaker than that for mammalian IRPs (5±2 nM).
• Leishmania IRE-binding activity is not induced by iron-depleted media, unlike mammalian IRPs.

Conclusions:

• Leishmania tarentolae possesses an IRE-binding protein, indicating that RNA-binding proteins involved in iron regulation are ancient.
• The identified Leishmania protein represents a potentially early form of IRP, offering insights into the evolution of RNA-binding domains.
• The lack of iron-dependent induction in Leishmania suggests divergence in regulatory pathways compared to vertebrates.