Trans-spliced mRNA products produced from circRNA expression vectors

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Expression

Background:

• Circular RNA (circRNA) expression vectors are crucial tools for identifying RNA sequences with internal ribosome entry site (IRES) activity.
• Accurate characterization of IRES elements is vital for understanding gene regulation and translation initiation.

Purpose of the Study:

• To investigate potential artifacts in circRNA expression vector-based IRES discovery.
• To identify alternative RNA splicing events that may confound IRES activity screens.

Main Methods:

• Development of a novel circRNA expression vector series for IRES screening.
• Analysis of pre-mRNA splicing patterns and resulting mRNA products.
• Characterization of trans-spliced mRNA products and their open reading frames.

Main Results:

• Evidence of trans-spliced mRNAs generated from embedded promoter sequences within circRNA vectors.
• These trans-spliced products can erroneously mimic authentic circRNAs.
• The observed trans-splicing events lead to false-positive signals in IRES discovery screens.

Conclusions:

• CircRNA expression vectors may produce artifacts that interfere with accurate IRES activity assessment.
• Trans-splicing events represent a significant confounding factor in circRNA-based IRES screens.
• Results emphasize the need for orthogonal validation methods beyond circRNA vectors for IRES discovery.