Enhancing mRNA Interactions by Engineering the Arc Protein with Nucleocapsid Domain

Area of Science:

• Neuroscience
• Molecular Biology
• Virology

Background:

• Activity-regulated cytoskeleton-associated protein (Arc) forms virus-like capsids for intracellular mRNA transport.
• HIV-1 Group-specific Antigen (Gag) binds viral mRNA via its Nucleocapsid (NC) domain.
• Mammalian Arc lacks an NC domain, leaving its mRNA binding interactions unclear.

Purpose of the Study:

• To investigate the direct mRNA binding interactions of rat Arc.
• To explore the role of the HIV-1 NC domain in Arc-mediated mRNA binding.
• To assess the influence of Arc's N-terminal domain-C-terminal assembly (NTD-CA) on mRNA binding.

Main Methods:

• In vitro binding assays were performed using rat Arc (rArc) and modified rArc constructs with rat Arc 5' UTR (A5U), HIV-1 5' UTR (H5U), and GFP mRNAs.
• Binding affinities were quantified and compared between different protein-mRNA combinations.
• The contribution of the HIV-1 Nucleocapsid (NC) domain and rArc's NTD-CA was evaluated.

Main Results:

• Native rat Arc exhibited weak, non-specific binding to A5U, H5U, and GFP mRNAs.
• Fusion of the HIV-1 NC domain to rArc significantly enhanced binding to H5U and A5U.
• The rArc NTD-CA domain was found to facilitate mRNA binding by the retroviral NC domain.

Conclusions:

• Mammalian Arc and HIV-1 Gag share conserved mechanisms for protein-mRNA interactions.
• The NTD-CA domain of Arc plays a crucial role in enhancing mRNA binding.
• Findings suggest potential for novel mRNA delivery systems by combining Gag NTD-CA with retroviral NC and UTRs.