Abstract A023: RNA secondary structures mediate circular RNA stability and function
Authors: Chack Aaron C , Harrison Emily B , Aufgebauer Caroline J , Boerneke Mark A , Irving Patrick S , Faison Edgar M , Zhao Jingyu , Zhang Qi , Weeks Kevin M , Pecot Chad V 
Published: November 2024

Abstract
Our group recently discovered that the circular RNA (circRNA), CDR1as, promotes lung cancer metastasis in part through the stabilization of the coding gene, CDR1. The purpose of this study was to identify intramolecular RNA secondary structures of CDR1as and determine whether there is a functional relationship between the CDR1as secondary structure and non-small cell lung cancer (NSCLC) metastasis. We employed the chemical probing approach selective 2’- hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to experimentally inform structure predictions of CDR1as. The SHAPE-MaP profiling experiments revealed that CDR1as is remarkably unstructured overall, with an exception of a highly probable and consistent stem-loop structure embedding the CDR1as backsplice junction (BSJ). Additionally, BSJ-embedding secondary structures were identified in all other mammalian circRNAs studied in a similar chemical probing experiment. To evaluate the role of the BSJ structure on CDR1as expression, we sterically disrupted the BSJ structure using a chemically modified, inert antisense oligonucleotide (BSJ-ASO) lacking a gapmer in order to block RNAse H activity. After transfection of the structure disrupting BSJ-ASO into several cell lines, we observed a 90-95% reduction of CDR1as expression by the BSJ-ASO by 24 hours. Furthermore, the BSJ-ASO significantly inhibited cancer cell proliferation and spheroid formation in several NSCLC models. Strikingly, using RNA stability assays we observed that the BSJ-ASO caused dramatic reductions in CDR1as levels within 5 minutes of transfection, suggesting a possibly hydrolytic process. A functional screen of silencing curated CDR1as RNA-binding proteins failed to rescue the loss of CDR1as expression following BSJ-ASO treatment. We next tested whether an RNA-catalyzed mechanism of action is possible. By incubating total RNA with the BSJ-ASO in buffers of varying magnesium chloride concentrations in vitro, we observed a dramatic decrease in CDR1as levels similar to that of the BSJ-ASO cellular transfection experiments, suggestive of ribozyme-like activity. Interestingly, use of a primer mapping approach indicated two putative cut sites within CDR1as. Further chemical probing following treatment with the BSJ-ASO revealed a new, highly probable secondary structure upstream of the BSJ, which appears similar to a hairpin ribozyme when modeled two-dimensionally. Taken together, our work reveals that intramolecular RNA secondary structures can have critical roles in maintaining circRNA cellular stability and function, and strategic steric disruption of these structures may induce circRNA self-cleavage.
Citation Format: Aaron C Chack, Emily B Harrison, Caroline J Aufgebauer, Mark A Boerneke, Patrick S Irving, Edgar M Faison, Jingyu Zhao, Qi Zhang, Kevin M Weeks, Chad V Pecot. RNA secondary structures mediate circular RNA stability and function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A023.