Gerst's lab
As mRNA localization is likely to play a critical role in protein localization in eukaryotic cells, we have initiated a genome-wide project to individually tag all endogenous nuclear-encoded mRNAs in yeast cells in order to determine whether there are specific patterns of localization. Spatial mapping of mRNAs in living cells defines the "mRNA locome". Thus far, we have devised and tested the technology necessary to allow for chromosomal tagging of individual yeast genes via homologous recombination. For each gene we use a PCR-based approach, which employs a single pair of oligonucleotides to amplify an integration construct which is integrated into the genome between the coding region and 3'-UTR. Integration places binding sites for a viral RNA binding protein between these elements and, upon co-expression of a fluorescent protein-tagged form of the viral protein, allows for localization of the mRNA to be detected using fluorescence microscopy. Currently, we are using this methodology to demonstrate the role of mRNA localization in the establishment and maintenance of cell polarity, as well as to define the mRNA locome for peroxisomal proteins. These important proofs of concept will pave the way for an expanded large-scale analysis of mRNA localization in yeast and, eventually, mammalian cells.
A. SEC4 mRNA and protein localization in yeast. mRNA encoding the Sec4 GTPase, which confers secretory vesicle fusion with the plasma membrane, was visualized in live cells using a construct expressing RFP-SEC4 mRNA having binding sites for a viral RNA binding protein. When co-expressed with the RNA binding protein fused to GFP, SEC4 mRNA localized to the incipient bud early in the cell cycle. RFP-Sec4 protein generated from the mRNA co-localized with GFP, demonstrating bud-specific translation of the mRNA. A second (untranslated) mRNA granule is apparent in the mother cell, representing new mRNA being readied in the nucleus for the next round of cell division. The results show that mRNA encoding a polarity factor is exported to the bud and results in local translation of the protein therein. This ensures localization of the polarity factor at the site of polarization.
B. OXA1 mRNA and protein localization in yeast. mRNA encoding a mitochondrial protein, Oxa1, was visualized in live yeast using OXA1-RFP mRNA bearing binding sites for an RNA binding protein. GFP fluorescence marks the site of OXA1-RFP mRNA localization, while RFP fluorescence marks the mitochondria. Thus, both OXA1-RFP mRNA and Oxa1-RFP protein appear to co-localize at mitochondria.
C. PEX3 mRNA localization to the ER in yeast. mRNA encoding a peroxisome assembly factor, Pex3, was visualized in yeast using PEX3 mRNA bearing binding sites for an RNA binding protein. GFP fluorescence marks the site of PEX3 mRNA localization, while Sec63-RFP fluorescence marks the ER. Thus, PEX3 mRNA is targeted to the ER. Since Pex3 protein first appears on the ER before reaching peroxisomes, our work suggests that it is first translated on ER membranes.
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