ASH1 mRNA pre-localizaton ribonucleoprotein assembly

Scripps Research Institute, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant) Messenger RNA (mRNA) localization is a widespread mechanism by which cells spatially restrict the synthesis of an encoded protein, and functions as an initial step in specifying asymmetric cell fates. Specific localized mRNAs commonly participate in ribonucleoprotein (RNP) complexes that are transported as cargo by motor proteins along cytoskeletal elements to their cellular destination. How mRNA localization RNPs assemble is largely unknown. In Saccharomyces cerevisiae, the ASH1 mRNA localization RNP is asymmetrically segregated to the budding daughter cell, where Ash1p regulates mating type. Three proteins--She2p, She3p and the unconventional myosin Myo4p--are known to be essential for ASH1 localization in vivo. I wish to understand mechanistically the biochemical and biophysical interactions that promote the assembly of this functional RNP. We expect the assembly of the ASH1 RNP to be regulated so that only fully formed RNPs are localized. To understand the interactions that promote complex assembly, I will determine the oligomerization state and bimolecular binding constants of the RNP components, and determine the influence of a third RNP component on the possible cooperativity of complex assembly. My goal is to reconstitute the ASH1 RNP in vitro using purified components, and compare the requirements of in vitro assembly to what is known about the RNP in vivo. My work will provide insight into the mechanisms of ribonucleoprotein assembly, and will begin to uncover the biochemical basis of this common developmental mechanism.