A sequential binding mechanism for 5' splice site recognition and modulation for the human U1 snRNP

Area of Science:

• Molecular Biology
• RNA Splicing
• Drug Discovery

Background:

• Splice site recognition is crucial for accurate transcriptome definition.
• Drugs like risdiplam and branaplam can alter 5' splice site (5'SS) recognition by U1 snRNP.
• Understanding the mechanism of 5'SS modulation is key for therapeutic development.

Purpose of the Study:

• To elucidate the sequential binding mechanism of branaplam in modulating -1A bulged 5'SS.
• To investigate how branaplam interactions lead to the formation of stable U1 snRNP/5'SS complexes.

Main Methods:

• Ensemble kinetic measurements.
• Colocalization single-molecule spectroscopy (CoSMoS).

Main Results:

• A sequential binding mechanism for branaplam at -1A bulged 5'SS was determined.
• Branaplam binds to the U1 snRNP/U1-C complex only after U1 snRNP engages with the 5'SS.
• Reversible binding interactions result in the formation of long-lived U1 snRNP/5'SS complexes.

Conclusions:

• Branaplam targets a ribonucleoprotein complex, not just an RNA duplex.
• The drug's mechanism relies on fundamental aspects of 5'SS recognition.
• This study provides a mechanistic basis for 5'SS modulators with therapeutic potential.