Roquin exhibits opposing effects on RNA stem-loop stability through its two ROQ domain binding sites

Area of Science:

• Structural biology
• Molecular biology
• Biophysics

Background:

• Posttranscriptional control relies on dynamic interactions between mRNA and regulatory proteins.
• Obtaining mechanistic insights into these interactions is challenging due to their dynamic nature.

Purpose of the Study:

• To analyze the interaction between Roquin's ROQ domain and the Ox40 mRNA 3'UTR using a combination of techniques.
• To elucidate the folding mechanisms of RNA structures and the binding kinetics of Roquin.

Main Methods:

• Single-molecule force spectroscopy (SMFS) to probe RNA folding and protein interactions.
• NMR spectroscopy to complement structural and dynamic insights.
• Integrated microfluidics for real-time binding kinetics extraction.

Main Results:

• Cooperative folding of the constitutive decay element (CDE) and complex folding pathways for the alternative decay element (ADE).
• Opposing effects of Roquin's A-site and B-sites on ADE structure and stability.
• Identification of RNA motif nature and Roquin subdomain plasticity as key determinants for RNA recognition.

Conclusions:

• Roquin's dual-function RNA-binding domain employs distinct mechanisms for target RNA recognition.
• The interplay between RNA structure and Roquin subdomains dictates mRNA decay specificity.
• This study provides a model for understanding Roquin-RNA complex formation and function.