Planta

, Volume 231, Issue 6, pp 1261–1276

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

  • Andrew J. Crofts
  • Naoko Crofts
  • Julian P. Whitelegge
  • Thomas W. Okita
Original Article

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 

Abbreviations

CDS

Coding sequence

ER

Endoplasmic reticulum

hnRNP

Heterogeneous nuclear ribonucleoprotein

IP

Immunoprecipitation

RBP

RNA binding protein

PB-ER

Protein body ER

RRM

RNA recognition motif

UTR

Untranslated region

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Andrew J. Crofts
    • 1
    • 2
  • Naoko Crofts
    • 1
    • 2
  • Julian P. Whitelegge
    • 3
  • Thomas W. Okita
    • 1
  1. 1.Institute of Biological ChemistryWashington State UniversityPullmanUSA
  2. 2.Akita International University, International Liberal Arts ProgramAkita-cityJapan
  3. 3.The Pasarow Mass Spectrometry Laboratory, NPI-Semel InstituteUCLA School of MedicineLos AngelesUSA

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