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

• Published in: Planta Vol. 231; no. 6; pp. 1261 - 1276
• Main Authors: Crofts, Andrew J, Crofts, Naoko, Whitelegge, Julian P, Okita, Thomas W
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.05.2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Agriculture; Baits; Base Sequence; Biological and medical sciences; Biomedical and Life Sciences; Carrier proteins; Cross-Linking Reagents - pharmacology; Cytoskeleton - metabolism; Ecology; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - radiation effects; Forestry; Fundamental and applied biological sciences. Psychology; Gels; Gene Expression Regulation, Plant - drug effects; Gene Expression Regulation, Plant - radiation effects; Glutens - genetics; Glutens - metabolism; Heterogeneous nuclear ribonucleoproteins; Heterogeneous-Nuclear Ribonucleoproteins - metabolism; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Intracellular Membranes - radiation effects; Life Sciences; Mass spectrometry; Messenger RNA; Microtubules - drug effects; Microtubules - metabolism; Microtubules - radiation effects; Molecular Sequence Data; Nucleic Acid Conformation; Open Reading Frames - genetics; Original Article; Oryza - drug effects; Oryza - embryology; Oryza - genetics; Oryza - metabolism; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Sciences; Prolamins - genetics; Prolamins - metabolism; Protein Binding; Proteins; Rice; RNA; RNA binding proteins; RNA Transport - drug effects; RNA Transport - radiation effects; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Seeds - drug effects; Seeds - genetics; Seeds - metabolism; Seeds - radiation effects; Storage proteins; Subcellular Fractions - drug effects; Subcellular Fractions - metabolism; Subcellular Fractions - radiation effects; Three prime untranslated regions; Ultraviolet Rays; Untranslated regions; RNA localization; Endoplasmic reticulum; Storage proteins; Rice; RNA binding proteins; Monocotyledones; Storage protein; RNA binding protein; Plant biology; Identification; Cereal crop; Oryza sativa; Binding protein; Messenger RNA; Gramineae; Seed; Angiospermae; Cytoskeleton; Isolation; Spermatophyta; Localization; Prolamin
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-010-1125-x

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.