Dissecting RNA selectivity mediated by tandem RNA-binding domains

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNA-protein interactions are crucial for gene regulation.
• Multidomain RNA-binding proteins (RBPs) often exhibit high RNA specificity, but their domain interplay in target selection is poorly understood.

Purpose of the Study:

• To investigate the interplay of RNA-binding domains within Musashi-1, Musashi-2, and unkempt proteins.
• To determine how protein intrinsic specificities and cellular context influence RBP target selection and regulation.

Main Methods:

• Massively parallel in vitro binding assays using random and natural RNA sequences.
• Large-scale competition assays to analyze RBP binding affinities and specificities.
• Integration of in vitro binding data with in-cell RNA-binding and regulation studies.

Main Results:

• Individual domains within RBPs display distinct affinities, specificities, and motif spacing preferences.
• Protein-protein competition assays reveal how specificities and affinities shape competitive binding.
• Cellular context is critical for directing RBPs to specific transcript regions, complementing intrinsic specificities.

Conclusions:

• RBP target selection is a complex process influenced by both intrinsic protein properties and cellular environment.
• Evolutionarily conserved RNA regions are high-affinity targets for multiple RBPs.
• Combined in vitro and in-cell studies are essential for comprehensive RBP profiling and understanding RNA-protein interactions.