Abstract
RNA binding proteins (RBPs) play an important role in mRNA metabolism including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements, or zipcodes. Putative trans-acting RBPs that recognize prolamine RNA zipcodes required for restricted localization to protein body-ER have previously been identified. Here, we describe the identification of RBP-P using a Northwestern blot approach as an RBP that recognizes and binds to glutelin zipcode RNA, which is required for proper RNA localization to cisternal-ER. RBP-P protein expression coincides with that of glutelin during seed maturation and is localized to both the nucleus and cytosol. RNA-immunoprecipitation and subsequent RT-PCR analysis further demonstrated that RBP-P interacts with glutelin RNAs. In vitro RNA–protein UV-crosslinking assays showed that recombinant RBP-P binds strongly to glutelin mRNA, and in particular, 3′ UTR and zipcode RNA. RBP-P also exhibited strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in mRNA splicing. Overall, these results support a multifunctional role for RBP-P in glutelin mRNA metabolism, perhaps in nuclear pre-mRNA splicing and cytosolic localization to the cisternal-ER.
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Acknowledgments
We thank Tin Yan Lau and Sethu Sivasubramanian for their technical assistance. Mass spectrometry analysis of samples was done by the University of Idaho Environmental Biotechnology Institute. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant No. 21380008 to T.K.) and by the National Science Foundation (Grant Nos. DBI–0605016, IOS–1021699, and Intergovernmental Personnel Act funds to T.W.O.). A.J.C. is grateful for financial support from the Japanese Society for the Promotion of Science in the form of a Grant-in-Aid for Young Scientists and to Akita Prefectural University for providing access to equipment. Preparation of the manuscript took placed while T.W.O. was on IR/D as an Intergovernmental Personnel Act (IPA) rotator at the National Science Foundation.
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Kelly A. Doroshenk and Li Tian have contributed equally to this work.
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Doroshenk, K.A., Tian, L., Crofts, A.J. et al. Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization. Plant Mol Biol 85, 381–394 (2014). https://doi.org/10.1007/s11103-014-0191-z
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DOI: https://doi.org/10.1007/s11103-014-0191-z