Abstract

Localized mRNA translation is involved in cell-fate determination, polarization and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. To facilitate the study of mRNA localization in yeast, we developed a simple method (m-TAG) for PCR -based chromosomal gene tagging that uses homologous recombination to insert binding sites for the RNA -binding MS2 coat protein (MS2-CP) between the coding region and 3’-untranslated region of any yeast gene. Upon co-expression of MS2 -CP fused with GFP , specific endogenously expressed mRNAs can be visualized in vivo. This method allows for the easy examination of mRNA localization using fluorescence microscopy and leaves the yeast cells amenable for further genetic analysis. We used m-TAG to determine the localization of different sets of mRNAs encoding proteins involved in different cellular processes, and found three distinct patterns of mRNAs encoding peroxisomal proteins localization: peroxisome-associated; ER-associated; and neither peroxisome nor ER-associated. This is the first time that mRNA was shown to be localized to peroxisomes. In addition, we tagged and localized mRNAs encoding actin cytoskeleton proteins and found that several mRNAs (e.g. MYO2 and MYO4 ) localized to the bud tip and to the bud neck. And finally, we examined the localization of mRNAs encoding autophagic proteins and found that ATG9 mRNA localized to both peripheral and nuclear ER, while ATG8 mRNA was found mostly on peripheral ER and on the vacuole. We also found that ATG8 mRNA localization is altered upon growth conditions and 3’UTR removal. The m-TAG method is now being used by others (in the lab, as well as other groups) and is an excellent tool for studying mRNA trafficking in yeast.