Abstract
One mechanism by which cells post-transcriptionally regulate gene expression is via intercellular and intracellular sorting of mRNA. In Saccharomyces cerevisiae, the localization of ASH1 mRNA to the distal tip of budding cells results in the asymmetric sorting of Ash1p to daughter cell nuclei. Efficient localization of ASH1 mRNA depends upon the activity of four cis-acting localization elements and also upon the activity of trans-factors She2p, She3p, and Myo4p. She2p, She3p, and Myo4p have been proposed to form an ASH1 mRNA localization particle. She2p directly and specifically binds each of the four ASH1 cis-acting localization elements, whereas She3p has been hypothesized to function as an adaptor by recruiting the She2p-mRNA complex to Myo4p, a type V myosin. The Myo4p-She3p-She2p heterotrimeric protein complex has been proposed to localize mRNA to daughter cells using polarized actin cables. Here we demonstrate that whereas the predicted Myo4p-She3p-She2p heterotrimeric complex forms in vivo, it represents a relatively minor species compared with the Myo4p-She3p complex. Furthermore, contrary to a prediction of the heterotrimeric complex model for ASH1 mRNA localization, ASH1 mRNA artificially tethered to She2p is not localized. Upon closer examination, we found that mRNA tightly associated with She2p is transported to daughter cells but is not properly anchored at the bud tip. These results are consistent with a model whereby anchoring of ASH1 mRNA requires molecular remodeling of the Myo4p-She3p-She2p heterotrimeric complex, a process that is apparently altered when mRNA is artificially tethered to She2p.
Highlights
MRNP.1 The mRNP localization complexes are dynamic structures undergoing molecular reorganization at various stages of the localization pathway (4 – 6)
A model for ASH1 mRNA localization was proposed that featured a Myop-She3p-She2p heterotrimeric complex, and ASH1 mRNA was hypothesized to associate with the heterotrimeric complex via She2p (24 –26)
The heterotrimeric complex model does not satisfactorily explain the observation that unlike endogenous She2p, which is uniformly distributed between mother and daughter cells, Myo4p and She3p are asymmetrically sorted to daughter cells dependent on associated mRNA [8, 22, 33, 37]
Summary
Ade, his, leu112, ura, trp1–1,ho, can100 Mata, ade, his, leu112, ura, trp, ho, can100, she2⌬ silent, MYO4-. Myc13:kanMX6 Mata, ade, his, leu112, ura, trp, ho, can100, she2⌬ silent, she3⌬. Silent, MYO4–3HA:kanMX6 Mata, ade, his, leu112, ura, trp, ho, can100, she2⌬ silent, she3⌬. Silent, MYO4-myc13:kanMX6 Mata, ade, his, leu112, ura, trp, ho, can100, SHE2-myc13:kanMX6 Mata, ade, his, leu112, ura, trp, ho, can100, SHE3-myc13:kanMX6 Mata, ade, his, leu112, ura, trp, ho, can100, MYO4-myc13:kanMX6. Closer inspection revealed that the heterotrimeric complex that is sufficient for transport of mRNA to daughter cells is insufficient for anchoring the mRNA at the bud tip. These results favor a model whereby anchoring of ASH1 mRNA requires reorganization of the Myo4p-She3p-She2p-ASH1 transport mRNP
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