Discovery of a mRNA Mitochondrial Localization Element in Saccharomyces Cerevisiae by Nonhomologous Random Recombination and In Vivo Selection

Abstract

In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3’ UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3’ end of various reporter mRNAs to induce mitochondrial localization. Sitedirected mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.