Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Nascent pre-mRNA 3 -end cleavage and polyadenylation (C/P) is a critical process regulated by protein-RNA interactions.
• The Human CSTF2 protein's RNA recognition motif (RRM) binds U-rich sequences, influencing C/P.
• A previously identified CSTF2 RRM disease mutant (CSTF2^D50A) alters RNA binding kinetics and leads to alternative polyadenylation.

Purpose of the Study:

• To investigate the role of electrostatic interactions in the thermodynamics and kinetics of CSTF2 RRM-RNA binding.
• To elucidate the downstream consequences of these interactions on C/P regulation.
• To understand the dynamic nature of RRM-RNA interactions and their functional implications.

Main Methods:

• Site-directed mutagenesis of the CSTF2 RRM.
• Nuclear Magnetic Resonance (NMR) spectroscopy.
• Biophysical assays (e.g., calorimetry, kinetics).
• In vivo C/P assays.

Main Results:

• Electrostatic attraction is the primary force driving CSTF2 RRM binding to U-rich RNA sequences.
• RNA binding involves an enthalpy-entropy compensation mechanism, modulated by protein dynamics on the pico- to nanosecond timescale.
• Dynamic binding allows the RRM to accommodate diverse nuclear RNA sequences.
• A balance exists between rapid, high-affinity RNA binding and efficient, accurate C/P.

Conclusions:

• The surface charge of the CSTF2 RRM is crucial for effective RNA binding.
• The interplay between RRM dynamics, RNA binding affinity, and C/P efficiency is vital for proper gene expression regulation.
• These findings provide insights into the molecular mechanisms governing pre-mRNA processing and its regulation.