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 can fail to digest >20% of 
all highly expressed linear RNAs, but these shortcomings can largely be 
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 polyadenylated 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.