Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds.
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.
Crofts
Andrew J
AJ
Institute of Biological Chemistry, Washington State University, Clark Hall, Room #299, 100 Dairy Road, Pullman, WA 99164-6340, USA. acrofts@aiu.ac.jp
Crofts
Naoko
N
Whitelegge
Julian P
JP
Okita
Thomas W
TW
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000528:radiation effects; D005983:Glutens / Q000235:genetics / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames / Q000235:genetics; D012275:Oryza / Q000187:drug effects / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055315:Prolamins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D034443:RNA Transport / Q000187:drug effects / Q000528:radiation effects; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012639:Seeds / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010940:Plant Proteins; D055315:Prolamins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D005983:Glutens