In vitro selection of translational regulatory elements

Untranslated regions (UTRs) of mRNAs carry various kinds of translational regulatory elements; however, our knowledge of them is still limited. We created an in vitro selection system that allows us to make a systematic enrichment of the sequences that alter translation efficiency (SESTRE) in any given mRNA and translation system. This method consists of the introduction of random nucleotide sequences into the UTRs of given mRNAs, followed by translation, size fractionation of the polyribosomes, and reverse transcription and PCR amplification (RT-PCR), with repeated cycles of these steps to enrich highly or poorly translatable mRNAs. With this experimental method, we examined how and where translational enhancer motifs emerge on mRNAs using the in vitro translation systems of wheat germ extract. The results indicate that the translational enhancers differentially emerge in response to the presence or absence of the 5′ cap. Interestingly, the translational enhancers that activate cap-independent translation evolved more readily in the 3′ UTR than in the 5′ UTR in wheat germ extract. This SESTRE method should be a powerful tool with which to improve the translational efficiency of given mRNAs in given translation systems and to investigate the structure-function relationship of eukaryotic mRNAs underlying translational control.