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

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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.

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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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Acknowledgments

This work was supported by National Science Foundation Grants No. IBN-0235140 and IOB-0544469.

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Author notes

  1. Andrew J. Crofts & Naoko Crofts

    Present address: Akita International University, International Liberal Arts Program, 193-2 Okutsubakidai, Yuwa-Tsubakigawa, Akita-city, 010-1292, Japan

Authors and Affiliations

  1. Institute of Biological Chemistry, Washington State University, Clark Hall, Room #299, 100 Dairy Road, Pullman, WA, 99164-6340, USA

    Andrew J. Crofts, Naoko Crofts & Thomas W. Okita

  2. The Pasarow Mass Spectrometry Laboratory, NPI-Semel Institute, UCLA School of Medicine, Los Angeles, CA, 90024-1759, USA

    Julian P. Whitelegge

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  1. Andrew J. Crofts
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  2. Naoko Crofts
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  4. Thomas W. Okita
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Corresponding author

Correspondence to Thomas W. Okita.

Additional information

A. J. Crofts and N. Crofts contributed equally to this work.

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Crofts, A.J., Crofts, N., Whitelegge, J.P. et al. Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds. Planta 231, 1261–1276 (2010). https://doi.org/10.1007/s00425-010-1125-x

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