Unraveling the Bivalent and Rapid Interactions Between a Multivalent RNA Recognition Motif and RNA: A Kinetic...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Musashi-1 (MSI1) is a key regulator of gene expression through RNA binding.
• Understanding the kinetics of MSI1-RNA interactions is crucial for deciphering its regulatory mechanisms.

Purpose of the Study:

• To characterize the binding kinetics of Musashi-1 (MSI1) and its variants to RNA.
• To elucidate the contribution of individual and tandem RNA recognition motifs (RRMs) to MSI1-RNA binding affinity and specificity.
• To develop a novel method for quantifying complex bivalent interactions.

Main Methods:

• Surface plasmon resonance (SPR) biosensor analysis was employed to study MSI1-RNA interactions.
• Truncated MSI1 variants with single (RRM1, RRM2) and tandem (RRM1-RRM2) RNA recognition motifs were engineered.
• SPR experiments involved injecting MSI1 variants over immobilized RNA sequences with varying lengths and binding motifs.

Main Results:

• Individual RRMs exhibited 1:1 binding kinetics with RNA, while the tandem RRM variant showed rapid, bivalent interactions.
• A new quantification method revealed that tandem RRMs significantly increase the residence time of MSI1 on RNA compared to single RRMs.
• MSI1 bivalency enhanced specificity for RNA with single UAG motifs over those with double UAG motifs or hairpin structures.
• Substitution of UAG with CAG motifs reduced individual RRM affinity, an effect strongly amplified in the bivalent MSI1 interaction.

Conclusions:

• Tandem RRMs in MSI1 contribute to enhanced RNA binding stability and specificity.
• Bivalency is a critical factor for MSI1's specific recognition of RNA targets.
• The findings provide insights into the functional significance of multiple RRMs in RNA-binding proteins.