Circular versus linear RNA topology: different modes of RNA–RNA interactions in vitro and in human cells

ABSTRACT

Circular RNA is progressively reported to occur in various species including mammals where it is thought to be involved in the post-transcriptional regulation of gene expression, partly via interactions with microRNA. Here, we asked whether the circular topology causes functional differences to linear forms when interacting with short RNA strands in vitro and in human cells. Kinetic studies with human bladder cancer-derived synthetic circular RNA versus linear transcripts, respectively, with short oligoribonucleotides showed similar association rates for both topologies. Conversely, a substantial topology-related difference was measured for the activation entropy and the activation enthalpy of RNA–RNA annealing. This finding strongly indicates a significant difference of the mechanism of RNA–RNA interactions. To investigate whether these characteristics of circular RNA are biologically meaningful we performed transient transfection experiments with a microRNA-regulated expression system for luciferase in bladder cancer-derived cells. We co-transfected linear or circular RNA containing one microRNA binding site for the target-suppressing microRNA mlet7a. Here, the circular isoform showed a strongly increased competition with microRNA function versus linear versions. In summary, this study suggests novel topology-related characteristics of RNA–RNA interactions involving circRNA in vitro and in living cells.

KEYWORDS:

• Circular RNA
• RNA-RNA annealing
• RNA kinetics
• RNA structure
• RNA thermodynamics
• microRNA
• mlet7a
• living cells

Acknowledgments

We cordially thank Rosel Kretschmer-Kazemi Far for helpful discussions and for carefully reading this manuscript, Patrick Lamm for initial support, Evelyn Knappe, and Fenja Fahrig for excellent assistance with the synthesis of circular RNA and for initial studies on RNA–RNA complex formation. We are also grateful to Jana Kochmann and Moritz Lübcke for support with RNA detection studies.

Disclosure statement

No conflict-of-interest declared.

Supplementary material

Supplemental data for this article can be accessed here