Original Article

Planta

, Volume 231, Issue 6, pp 1261-1276

First online:

Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds

  • Andrew J. CroftsAffiliated withInstitute of Biological Chemistry, Washington State UniversityAkita International University, International Liberal Arts Program
  • , Naoko CroftsAffiliated withInstitute of Biological Chemistry, Washington State UniversityAkita International University, International Liberal Arts Program
  • , Julian P. WhiteleggeAffiliated withThe Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute, UCLA School of Medicine
  • , Thomas W. OkitaAffiliated withInstitute of Biological Chemistry, Washington State University Email author 

Rent the article at a discount

Rent now

* Final gross prices may vary according to local VAT.

Get Access

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 upon 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 cis-localization sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the cis-localization RNA bait sequence led to the capture of a much larger number of proteins, 18 of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analyzed to date show good to excellent specificity for binding to cis-localization sequences over the control RNA bait. Temporal expression studies showed that steady state protein levels for one RNA binding protein, RBP-A, paralleled prolamine gene expression. Immunoprecipitation studies showed that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression. Using confocal immunofluorescence microscopy, RBP-A was found to be distributed to multiple compartments in the cell. In addition to the nucleus, RBP-A co-localizes with microtubules and is associated with cortical ER membranes. Collectively, these results indicate that employing a combination of in vitro binding and in vivo binding and localization studies is a valid strategy for the identification of putative prolamine mRNA binding proteins, such as RBP-A, which play a role in controlling expression of storage protein mRNAs in the cytoplasm.

Keywords

Endoplasmic reticulum Rice RNA binding proteins RNA localization Storage proteins