A spring-loaded grip-and-pull mechanism for stepwise RNA duplex unwinding by Xrn1

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Xrn1 is a key enzyme in eukaryotic RNA degradation and quality control.
• Its mechanism for unwinding structured RNA remains unclear.

Purpose of the Study:

• To investigate the role of specific arginine residues in Xrn1's duplex unwinding activity.
• To elucidate the mechanism of Xrn1's interaction with structured RNA substrates.

Main Methods:

• Site-directed mutagenesis to substitute arginine residues (R100, R101) with lysine.
• Enzyme activity assays to measure exonuclease function on various RNA substrates.
• Single-molecule Förster resonance energy transfer (smFRET) to observe unwinding dynamics.

Main Results:

• Mutations R100K and R101K significantly impaired Xrn1 exonuclease activity, particularly on structured substrates like RNA-DNA hybrids.
• R101K showed a more severe defect, indicating specific roles for these residues.
• smFRET revealed stepwise duplex unwinding, melting ~8-9 base pairs per step.
• Results suggest a charge-dependent mechanism involving electrostatic interactions for duplex destabilization.

Conclusions:

• Conserved arginine residues R100 and R101 are critical for Xrn1's ability to unwind structured RNA duplexes.
• These residues modulate Xrn1's processivity on complex substrates through charge-dependent interactions.
• The findings provide new insights into the mechanism of RNA duplex unwinding by Xrn1.