The chicken c-Jun 5′ untranslated region directs translation by internal initiation

Abstract

The 5′ untranslated region (UTR) of the chicken c-jun message is exceptionally GC rich and has the potential to form a complex and extremely stable secondary structure. Because stable RNA secondary structures can serve as obstacles to scanning ribosomes, their presence suggests inefficient translation or initiation through alternate mechanisms. We have examined the role of the c-jun 5′ UTR with respect to its ability to influence translation both in vitro and in vivo. We find, using rabbit reticulocyte lysates, that the presence of the c-jun 5′ UTR severely inhibits translation of both homologous and heterologous genes in vitro. Furthermore, translational inhibition correlates with the degree of secondary structure exhibited by the 5′ UTR. Thus, in the rabbit reticulocyte lysate system, the c-jun 5′ UTR likely impedes ribosome scanning resulting in inefficient translation. In contrast to our results in vitro, the c-jun 5′ UTR does not inhibit translation in a variety of different cell lines suggesting that it may direct an alternate mechanism of translational initiation in vivo. To distinguish among the alternate mechanisms, we generated a series of bicistronic expression plasmids. Our results demonstrate that the downstream cistron, in the bicistronic gene, is expressed to a much higher level when directly preceded by the c-jun 5′ UTR. In addition, inhibition of ribosome scanning on the bicistronic message, through insertion of a synthetic stable hairpin, inhibits translation of the first cistron but does not inhibit translation of the cistron downstream of the c-jun 5′ UTR. These results are consistent with a model by which the c-jun message is translated through cap independent internal initiation.