Engineering back-splicing-like sites for enhanced linear RNA expression

The formation of linear RNAs and circRNAs from pre-RNAs is a competitive process. The factors that determine which type of RNA molecule is formed include the sequence of the pre-mRNA, the presence of regulatory elements, and the activity of RNA-binding proteins. In this study, multiple circRNAs were formed by introducing a Luc expression vector or in vitro transcribed Luc RNAs into three different cell lines: HepG2, BmN, and CIK cells. These models are widely used for studying hepatocellular carcinoma, silkworm molecular biology, and Ctenopharyngodon idellus-pathogen interactions. The existence of Luc-derived circRNAs was verified via reverse transcription‒PCR, Sanger sequencing, and reverse transcription‒rolling circle amplification. Sequence analysis of the circRNA junction sites revealed that the back splicing-like (BSL) sites of these Luc-circRNAs were different from the canonical splice sites. Mutations at BSL sites can significantly reduce the expression levels of Luc-derived circRNAs and increase the expression levels of Luc mRNA in cells. Furthermore, luciferase activity was significantly increased in BSL site mutation-transfected cells. In addition, the efficiency of circRNA formation was reduced, and the corresponding RNAs were increased in cells infected with recombinant baculoviruses with BSL site mutations. Furthermore, the silencing of circRNA formation-related protein genes significantly reduced the expression efficiency of Luc-derived circRNAs and significantly increased the expression level of Luc. These studies provide valuable insights into the regulation of BSL-mediated gene expression and contribute to the development of more efficient and targeted gene expression systems. KEY POINTS: • Exogenous genes that enter cultured cells can be spliced. • A change in the BSL site of an exogenous gene could improve its expression. • The regulation of BSL sites could be used to develop a method for efficient gene expression.