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Use of Alu Element Containing Minigenes to Analyze Circular RNAs
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Engineering back-splicing-like sites for enhanced linear RNA expression.

Xialing Chen1, Sufei Sun1, Kaimeng Li2

  • 1School of Life Sciences, Suzhou Medical College of Soochow University, Suzhou, 215123, China.

Applied Microbiology and Biotechnology
|October 16, 2025
PubMed
Summary

Introducing exogenous genes into cells can form circular RNAs (circRNAs) or linear RNAs. Modifying back splicing-like (BSL) sites in these genes enhances their expression, offering a new gene expression system.

Keywords:
Luc geneLuc-derived circRNAsBack-splicing-like site

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Area of Science:

  • Molecular Biology
  • RNA Biology
  • Gene Expression

Background:

  • Circular RNA (circRNA) and linear RNA formation from pre-RNA is a competitive process influenced by RNA sequence, regulatory elements, and RNA-binding proteins.
  • Exogenous gene expression in cultured cells involves complex splicing mechanisms.

Purpose of the Study:

  • To investigate the formation of circRNAs from exogenous luciferase (Luc) genes in different cell lines.
  • To analyze the role of back splicing-like (BSL) sites in the formation and expression of Luc-derived circRNAs.
  • To explore the potential of regulating BSL sites for developing efficient gene expression systems.

Main Methods:

  • Luciferase (Luc) expression vectors and in vitro transcribed Luc RNAs were introduced into HepG2, BmN, and CIK cells.
  • circRNA detection using reverse transcription–polymerase chain reaction (RT–PCR), Sanger sequencing, and reverse transcription–rolling circle amplification.
  • Mutation analysis of BSL sites and assessment of their impact on RNA expression and luciferase activity.
  • Silencing of circRNA formation-related protein genes.

Main Results:

  • Luc-derived circRNAs were successfully formed and detected in multiple cell lines.
  • BSL sites, distinct from canonical splice sites, were identified in Luc-circRNAs.
  • Mutations at BSL sites significantly reduced circRNA expression while increasing linear Luc mRNA levels and luciferase activity.
  • Recombinant baculoviruses with BSL site mutations showed reduced circRNA formation and increased RNA expression.
  • Silencing of key protein genes involved in circRNA formation decreased circRNA efficiency and increased Luc expression.

Conclusions:

  • Exogenous genes introduced into cultured cells can undergo splicing to form circRNAs.
  • Modifying BSL sites in exogenous genes can enhance their expression, offering a novel regulatory mechanism.
  • Regulation of BSL sites presents a promising strategy for developing efficient and targeted gene expression systems.