Abstract
Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5′ untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA.
Highlights
All eukaryotic mRNAs are co-transcriptionally capped with a m7G cap [1]
We showed that backsplicing and an internal ribosomal entry sites (IRESs) are required for NanoLuc production
RNA circles by a backsplicing event that is facilitated by complementary sequences upstream and downstream of the RNA sequence of interest included in the circle
Summary
All eukaryotic mRNAs are co-transcriptionally capped with a m7G cap [1]. This cap structure is required to protect mRNAs from exonucleases and degradation, and to enable translation initiation. The m7G cap is bound by the capbinding protein eukaryotic initiation factor (eIF) 4E, which binds to the scaffolding protein eIF4G [2,3]. Together, these two proteins along with the helicase eIF4A, form the initiation complex eIF4F, a key complex for translation initiation [4,5]. Many viruses initiate translation independent of the eIF4F complex. Instead, these viruses use RNA secondary structures known as internal ribosomal entry sites (IRESs) to recruit ribosomes to the viral RNA [6,7]. IRESs were initially discovered in poliovirus and encephalomyocarditis virus (EMCV) but were since found in a variety of viruses from different virus families [8,9,10]
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