A circular split nanoluciferase reporter for validating and screening putative internal ribosomal entry site elements

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Internal ribosomal entry sites (IRESs) are RNA elements that initiate cap-independent translation.
• While IRESs are common in viruses, their existence and function in mammalian mRNA remain debated.
• Existing bicistronic reporter assays for IRES discovery are susceptible to false positives from cryptic promoters or splicing sites.

Purpose of the Study:

• To develop a novel and more reliable assay for screening and validating IRES activity.
• To re-evaluate previously reported cellular IRESs using the new assay.

Main Methods:

• Development of a genetically encoded circular RNA reporter system utilizing a split nanoluciferase (nLuc) construct.
• Comparison of the circular split nLuc reporter assay with traditional bicistronic reporter assays.
• Screening of nine reported cellular IRES candidates using the circular split nLuc assay.

Main Results:

• The circular split nLuc reporter assay is less prone to false positives compared to bicistronic assays.
• Nine previously reported cellular IRESs exhibited minimal IRES activity when tested with the new assay.
• The circular split nLuc reporter provides a streamlined approach for IRES screening.

Conclusions:

• The circular split nLuc reporter assay is a robust and simplified tool for identifying and validating IRESs.
• The findings challenge the functional significance of several previously proposed cellular IRESs.
• This new method enhances the accuracy of IRES discovery in mammalian systems.