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

Abstract

Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (AMD1 tail). To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression governed by EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assay and western blotting to explore the effects of AMD1 tail and control sequences on reporter expression in dual and monocistronic reporter vectors.       Results:  With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. The decreased reporter activity that appears to be induced by the readthrough sequence occurs only in reporters containing EMCV IRES. Monocistronic reporters with the same readthrough context sequence exhibit only a modest reduction in reporter activity. Furthermore, in monocistronic vectors, the disproportionate reduction of reporter levels greatly increased when AMD1 tail was translated as a result of readthrough. Such readthrough-mediated reduction was not observed when AMD1 tail was substituted with unrelated sequences in agreement with our original hypothesis. Conclusions: While our findings provide little new information regarding the functional role of AMD1 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.