A Split NanoLuc Reporter Quantitatively Measures Circular RNA IRES Translation

Area of Science:

• Molecular Biology
• RNA Biology
• Translational Control

Background:

• Internal ribosomal entry sites (IRESs) are RNA structures enabling cap-independent translation.
• Current IRES assays, like the dicistronic luciferase assay, are quantitative but complex and time-consuming.
• Circular RNAs offer an alternative for IRES reporting but face challenges like low yield.

Purpose of the Study:

• To develop a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter for IRES activity.
• To validate the reporter's ability to measure IRES-mediated translation accurately.
• To assess IRES function under cellular stress and identify novel IRES elements.

Main Methods:

• Utilized a backsplicing circular RNA split GFP reporter system.
• Engineered a split NanoLuc reporter construct to quantify IRES-mediated translation.
• Assessed reporter expression under basal and cellular stress conditions.
• Tested the reporter with known and putative IRES sequences, including the Zika virus 5' UTR.

Main Results:

• NanoLuc expression was dependent on backsplicing and correct IRES orientation.
• The IRES-directed NanoLuc reporter remained stable or increased during cell stress, unlike a capped control.
• Detected NanoLuc expression from putative cellular IRESs and the Zika virus 5' UTR.

Conclusions:

• The developed split NanoLuc circular RNA reporter is a sensitive and quantitative tool for IRES analysis.
• This reporter system can effectively verify, identify, and quantify IRES-mediated translation.
• The reporter is valuable for studying IRES function under various conditions, including cellular stress and viral elements.