Functional analysis of the 5' untranslated region of the c-myc proto-oncogene

Previous reports suggested that the predominant c-myc 5'UTRs did not alter the translational efficiency of mRNA in vivo.;Expression of heterologous mRNAs fused to the principal c-myc 5'UTR (derived from the P2 transcript) in various cell lines confirmed that this element did not inhibit translation initiation. however, insertion of tihs sequence between the two cistrons of a dicistronic mRNA demonstrated that the 5' UTR stimulated the expression of the downstream cistron. Synthesis of the second cistron from the 5' UTR-containing dicistronic mRNA was shown to be independent of translation initiation at the upstream cistron start codon. In addition, the enhanced downstream cistron expression was shown to occur on intact dicistronic mRNAs. These data strongly suggest that the c-myc 5'UTR contains an internal ribosome entry segment capable of directing ribosomes to a site downstream of the 5' end of the mRNA.;Deletion analysis was used to define the minimum elment required for c-myc IRES-directed translation. These experiments demonstrated that IRES is located between nucelotides -396 and -57 of the human c-myc P2 5'UTR. Interestingly, the activity of the c-myc IRES may depend on limiting factors. Furthermore, internal initiation directed by the c-myc 5'UTR was very inefficient on dicistronic mRNAs lacking a nuclear origin. Thus, a prior nuclear event may also be necessary for c-myc IRES-driven translation initiation.;Finally, the effect of the c-myc 5' leader sequences was analyzed in rabbit reticulocyte lysate. The 5' UTR was unable to promote internal ribosome entry in this system. In fact, both the P2 and P1 sequences reduced the translational efficiency of mRNAs. Furthermore, the P2 5' UTR displayed a strong dependence on the 5' cap structure.;The 5' UTR may modulate c-myc protein synthesis through both the cap-dependent and internal initiation mechanism of translation.