ИСТИНА

eIF2D and its homologs MCTS1 and DENR are eukaryotic translation factors with unknown function. In mammalian translation reconstitution system both eIF2D and MCTS1/DENR dimer have an activity of tRNA stabilization in the P-site of 40S ribosomal subunits. This suggests a role in translation initiation, termination or recycling played by these proteins in vivo. In drosophila, the orthologous proteins were reported to be necessary for translation of uORF containing mRNAs. To dissect the function of eIF2D, MCTS1 and DENR further, we turned to yeast cell-free translation systems prepared from two S.cerevisiae strains, wt and the double knockout strain lacking both TMA64 (a EIF2D ortholog) and TMA20 (an MCTS1 orholog) genes. A set of luciferase encoding mRNAs with 5’ untranslated regions bearing different uORFs was prepared and translated in these in vitro systems. To check specificity, we performed rescue of Δtma64/Δtma20 lysate by addition of the recombinant human MCTS1/DENR dimer. We observed an enhanced translation of the uORF containing mRNAs in the absence of TMA64 and TMA20, in contrast to uORF-less ones. The effect required termination of the uORF translation before or shortly behind the luciferase start codon. It also depends on uORF length and a distance between uORF and the reporter coding sequence. We concluded that reinitiation rate is increased in the absence of TMA64 and TMA20. Thus, these proteins are involved in translation reinitiation. We hypothesize that they may operate to protect the post-termination ribosomes from acquiring novel initiation complex and thus inhibits unsanctioned translation reinitiation on eukaryotic mRNAs.