An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing...

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• Circular RNAs (circRNAs) are prevalent in eukaryotes, formed by covalently linked ends.
• RNase R, a 3'-5' exonuclease, is commonly used to enrich circRNAs by degrading linear RNAs.
• Standard RNase R protocols may incompletely digest certain linear RNAs, affecting circRNA isolation purity.

Purpose of the Study:

• To identify and overcome limitations in current RNase R-based methods for circular RNA enrichment.
• To improve the efficiency and accuracy of circular RNA isolation techniques.
• To investigate RNA structures that confer resistance to exonuclease degradation.

Main Methods:

• Investigated RNase R digestion efficiency on various linear RNAs, including structured RNAs and mRNAs.
• Introduced poly(A) tails to structured RNAs to assess their impact on RNase R degradation.
• Modified reaction buffers by replacing K+ with Li+ to disrupt G-quadruplex (G4) structures and observe RNase R activity.

Main Results:

• Standard RNase R protocols failed to digest over 20% of highly expressed linear RNAs.
• Poly(A) tail addition enabled efficient RNase R degradation of structured RNAs like snRNAs and histone mRNAs.
• RNase R stalled at G-rich sequences forming G-quadruplexes in mRNAs; Li+ addition facilitated complete degradation by resolving these structures.

Conclusions:

• Optimized protocols involving poly(A) tailing and Li+-containing buffers significantly improve linear RNA digestion by RNase R.
• These improvements enhance the biochemical enrichment of circular RNAs.
• The findings reveal RNA structural elements that inhibit exonuclease activity and provide methods to overcome this resistance.