A Dual-Selection System for Enhanced Efficiency and Fidelity of Circular RNA Overexpression

Circular RNAs (circRNAs) are essential regulators of cellular processes, but are challenging to study using traditional methods. Overexpression approaches, such as the use of linearized plasmids and viral vectors, often result in high rates of false-positive clones, where cells retain selection markers without expressing the target circRNA. This study addresses this limitation by developing a dual-selection cir-cRNA system designed to enhance the accuracy and reliability of circRNA overexpression. Our system integrates a fluorescent reporter gene upstream of the circRNA expression cassette, under a shared pro-moter, and a downstream antibiotic resistance marker, allowing for both antibiotic selection and flow cyto-metric cell-sorting to identify and enrich cells with genuine circRNA expression. We successfully incorporated this system into an inducible lentiviral vector for controlled overexpression in various cell types. The dual-selection circRNA system offers a significant advance for circRNA research and studies of other RNA species where accurate and reliable overexpression is essentialDawei Liu, Xing Wang, Yali Zhang, Shiyi Zuo, Bradley Chereda, Philip A. Gregory, Chun-Xia Zhao, and Gregory J. Goodal

On the rules of engagement for microRNAs targeting protein coding regions

OnlinePublMiRNAs post-transcriptionally repress gene expression by binding to mRNA 3 UTRs, but the extent to which they act through protein coding regions (CDS regions) is less well established. MiRNA interaction studies show a substantial proportion of binding occurs in CDS regions, however sequencing studies show much weaker effects on mRNA levels than from 3 UTR interactions, presumably due to competition from the translating ribosome. Consequently, most target prediction algorithms consider only 3 UTR interactions. However, the consequences of CDS interactions may have been underestimated, with the reporting of a novel mode of miRNA-CDS interaction requiring base pairing of the miRNA 3 end, but not the canonical seed site, leading to repression of translation with little effect on mRNA turnover. Using extensive reporter, western blotting and bioinformatic analyses, we confirm that miRNAs can indeed suppress genes through CDS-interaction in special circumstances. However, in contrast to that previously reported, we find repression requires extensive base-pairing, including of the canonical seed, but does not strictly require base pairing of the 3 miRNA terminus and is mediated through reducing mRNA levels. We conclude that suppression of endogenous genes can occur through miRNAs binding to CDS, but the requirement for extensive basepairing likely limits the regulatory impacts to modest effects on a small subset of targets.Sunil Sapkota, Katherine A. Pillman, B. Kate Dredge, Dawei Liu, Julie M. Bracken, Saba Ataei Kachooei, Bradley Chereda, Philip A. Gregory, Cameron P. Bracken, and Gregory J. Goodal