An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Xiao MS,Wilusz JE

doi

10.1093/nar/gkz576

subject

Has Abstract

pub_date

2019-09-19 00:00:00

pages

8755-8769

issue

16

eissn

0305-1048

issn

1362-4962

pii

5527976

journal_volume

47

pub_type

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