Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• High-throughput RNA sequencing identifies numerous circular RNAs (circRNAs).
• Alternative splicing generates circRNA splice variants with identical backsplice junctions, posing identification challenges.
• Understanding circRNA diversity is crucial for deciphering their distinct biological roles.

Purpose of the Study:

• To develop and validate a method for identifying full-length circRNA splice variants.
• To experimentally confirm computationally predicted circRNA variants.
• To investigate the functional implications of different circRNA splice variants.

Main Methods:

• Developed circRNA-Rolling Circle Amplification (circRNA-RCA) method.
• Utilized reverse transcription without RNase H activity.
• Employed PCR amplification with primers spanning the backsplice junction and upstream region.
• Sequenced PCR products to identify full-length circRNA sequences.

Main Results:

• Successfully identified and experimentally validated circRNA splice variants with identical backsplice junctions.
• Demonstrated that circRNA variants can be distinguished based on their full-length sequences.
• Predicted distinct associations of splice variants with RNA-binding proteins and microRNAs.

Conclusions:

• The circRNA-RCA method enables accurate identification of full-length circRNA sequences.
• This technique provides valuable insights into the functional diversity of circRNA splice variants.
• Experimental validation of circRNA variants is essential for understanding their individual biological impacts.