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

Overview

Internal ribosomal entry sites (IRESs) are sequences that can recruit the ribosome to promote translation, typically in an m7G cap-independent manner. IRESs are often found in 5' UTRs of positive-strand RNA viral genomes and drive translation of viral proteins. IRESs can also be found in mammalian transcriptomes where they mediate cap-independent translation of mRNAs. Discovery and characterization of both types of IRESs is important due to their ability to shed light on translation mechanisms and for use in therapeutic applications. However, current methods for screening IRES activity rely on a bicistronic reporter assay which require additional experiments to control for false positive results that derived from cryptic promoters and cryptic splicing. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and is therefore a more streamlined method for screening IRES activity. We use the circular split nLuc reporter to test putative cellular IRESs and compare viral IRESs. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs with reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay.