Overview

Asymmetric mRNA localization is a widely used mechanism to sort cell fate determinants in development. In budding yeast ASH1 mRNA localization to the growing bud leads to targeting of Ash1p to the daughter cell, a prerequisite for proper mating type switching. In a genetic screen 5 SHE genes required for ASH1 mRNA localization have been isolated. SHE1 is equivalent to MYO4 - a locus that encodes a myosin - suggesting a cytoskeleton-based, active transport mecha-nism. Functional characterization in turn has implied Myo4p as the motor protein, She2p as the ASH1 mRNA-binding protein, and She3p as adapter protein connecting She2p to Myo4p. Accessory factors such as Loc1p or Khd1p have been reported to function in ASH1 mRNA localization though they have not been identified in the genetic screen. To allow a detailed characterization of the ASH1-She RNP (ribo-nucleoprotein) complex, I initiated a tandem affinity purification protocol, using either She2p or Myo4p as bait protein. In either case I could purify the core She machinery together with ASH1 mRNA, arguing for the integrity of a single RNP. Furthermore, mass-spectrometric analysis identified a number of so far unknown proteins.

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9783639023138

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