ИСТИНА

m7G cap at the 5’ end of eukaryotic mRNAs is a key determinant of translational efficiency since it facilitates the first step of translation initiation - loading of ribosomes and initiation factors at the 5’end of mRNA. The ribosome with initiation factors then unwinds the secondary structures and moves along the 5’UTR to locate the initiation codon. It is currently believed that the cap is more essential for the translation of mRNAs with long and structured 5’UTRs. However, we demonstrate here that the long (577 nt) and highly structured 5’UTR of Apaf-1 mRNA can efficiently direct translation in vivo, and omission of m7G cap results in a less dramatic effect on its translational efficiency as compared to the control standard 5’UTRs of beta-globin or beta-actin (5-7 fold reduction vs 50-80 fold for the control). This difference is not accounted for by the presence of an IRES within the Apaf-1 5’ UTR since 1) the translation of the capped monocisrtonic mRNA directed by the Apaf-1 5’UTR is two orders of magnitude more efficient than that for the same 5’ UTR in a bicistronic position; 2) insertion of uAUG codons into the 5’UTR dramatically inhibits translation of not only capped, but also uncapped monocistronic mRNAs. We conclude that this 5’ UTR is scanned by the ribosomes from the 5’end irrespective of capping. Mutational analysis of Apaf-1 5’UTR shows that at least two of its structural modules are responsible for the reduced dependence of translation on the cap. Moreover, one of them individually possesses a reduced cap-dependence: if compared with short unstructured 5’UTRs, it directs translation less efficiently when it is capped, but more efficiently when it is uncapped. Mutation analysis of this fragment shows that deletion of Y-shaped stem-loop structure greatly increases dependence on the cap. Although the molecular mechanism of the reduced cap-dependence of Apaf-1 leader remains to be established, we propose that it can explain a relative resistance of Apaf-1 translation in vivo to apoptosis, the phenomenon which is currently explained by the existence of an IRES within the Apaf-1 5’UTR. Indeed, treatment of cells with etoposide, agent that causes apoptosis, and as a result inactivates cap-binding initiation factors (eIF4E is sequestered by 4E-BP and eIF4G is cleaved by a caspase) affects the 5’end dependent translation of mRNAs with standard 5’ leaders more dramatically than that directed by the Apaf-1 5’UTR.