Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES.

 Previously we reported the discovery of stop codon readthrough in mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed . To explain the severe suppression of reporters fused to tail we proposed a mechanism invoking ribosome queueing. In the original study, we tested this hypothesis, by placing the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Here we employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors.  With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to tail and occurs as long as the readthrough stop codon context is present at the end of the reporter's ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g., tail inhibitory effect. We further show by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of RNA levels.   While our findings provide little new information regarding the functional role of tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of readthrough permissive sequences and of IRES elements, though these require a separate investigation.