GSE130905
An improved method for circular RNA purification that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends
Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R fail to digest >20% of all highly expressed linear RNAs, but that these shortcomings can be easily overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases. Overall design: RNA-seq experiments: (1) Two replicates of HeLa cell derived RNA were treated with either RNase R or KCl containing buffer for 15 min. (2) Three replicates of HeLa cell derived RNA were treated by E-PAP followed by digestion with RNase R in LiCl containing buffer. The correspoding three controls were treated with LiCl containing buffer alone.