Abstract
The messenger RNA of the rice seed storage protein prolamine is targeted to the endoplasmic reticulum (ER) membranes surrounding prolamine protein bodies via a mechanism which is dependent on both RNA sorting signals and the actin cytoskeleton. In this study we have used an RNA bait corresponding to the previously characterized 5'CDS prolamine zipcode sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the zipcode RNA bait sequence captured a much larger number of proteins, eighteen of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analysed to date show good to excellent specificity for binding to zipcode over non-zipcode RNA bait. The protein expression profile of RBP-A, an hnRNP, closely follows prolamine gene expression and in addition to being present in nuclei, RBP-A also co-localizes with microtubules and is associated with cortical ER membranes. Immunoprecipitation studies show that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression.
