An Improved Model for Circular RNA Overexpression: Using the Actin Intron Reveals High Circularization Efficiency

Area of Science:

• Molecular Biology
• RNA Biology
• Biotechnology

Background:

• Traditional methods using the T4 td gene group 1 intron for circular sequence generation exhibit low circular RNA (circRNA) expression efficiency.
• Low efficiency hinders robust functional studies of circRNAs.

Purpose of the Study:

• To develop a novel, highly efficient method for expressing circular sequences, specifically circRNAs.
• To optimize circRNA expression by testing actin intron sequences of varying lengths.

Main Methods:

• Construction of circRNA expression plasmids using actin intron sequences (15-nt to 180-nt) and T4 intron.
• Introduction of plasmids into human (293T) and mouse (B16) cell lines.
• Assessment of circularization efficiency via junction detection, sequencing, and RNA pull-down assays followed by mass spectrometry (MS).

Main Results:

• Medium-length actin introns (60-nt to 100-nt) demonstrated significantly increased circularization efficiency.
• The 100-nt actin intron exhibited the highest efficiency across most tested conditions.
• Mass spectrometry identified potential splicing factors involved in the high circularization mechanism.

Conclusions:

• A new circRNA expression system utilizing optimized actin intron lengths offers high circularization efficiency.
• This improved system provides a more reliable platform for future circRNA functional research.
• The findings contribute to advancing the study of circRNAs and their biological roles.