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SUMMARY:Endogenous mRNA localization in living yeast - Liora Haim-Vilmovsk
 y\, Department of Molecular Genetics\, Weizmann Institute of Science\, Isr
 ael.
DTSTART:20100702T100000Z
DTEND:20100702T110000Z
UID:TALK25402@talks.cam.ac.uk
CONTACT:M. Madan Babu
DESCRIPTION:Localized mRNA translation is involved in cell-fate determinat
 ion\, polarization and morphogenesis in eukaryotes.  While various tools a
 re available to examine mRNA localization\, no easy and quick method has a
 llowed for the visualization of endogenously expressed mRNAs in vivo. To f
 acilitate 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 protei
 n (MS2-CP) between the coding region and 3’-untranslated region of any y
 east gene.  Upon co-expression of MS2-CP fused with GFP\, specific endogen
 ously expressed mRNAs can be visualized in vivo. This method allows for th
 e easy examination of mRNA localization using fluorescence microscopy and 
 leaves the yeast cells amenable for further genetic analysis. We used m-TA
 G to determine the localization of different sets of mRNAs encoding protei
 ns involved in different cellular processes\, and found three distinct pat
 terns of mRNAs encoding peroxisomal proteins localization: peroxisome-asso
 ciated\; ER-associated\; and neither peroxisome nor ER-associated. This is
  the first time that mRNA was shown to be localized to peroxisomes. In add
 ition\, we tagged and localized mRNAs encoding actin cytoskeleton proteins
  and found that several mRNAs (e.g. MYO2 and MYO4) localized to the bud ti
 p and to the bud neck. And finally\, we examined the localization of mRNAs
  encoding autophagic proteins and found that ATG9 mRNA localized to both p
 eripheral and nuclear ER\, while ATG8 mRNA was found mostly on peripheral 
 ER and on the vacuole. We also found that ATG8 mRNA localization is altere
 d upon growth conditions and 3’UTR removal. The m-TAG method is now bein
 g used by others (in the lab\, as well as other groups) and is an excellen
 t tool for studying mRNA trafficking in yeast.
LOCATION:Level 4 Cell Biology Seminar Room\, MRC Laboratory of Molecular B
 iology\, Cambridge\, UK
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