Zipcode RNA-Binding Proteins and Membrane Trafficking Proteins Cooperate to Transport Glutelin mRNAs in Rice Endosperm.

In rice (Oryza sativa) endosperm cells, mRNAs encoding glutelin and prolamine are translated on distinct cortical-endoplasmic reticulum (ER) subdomains (the cisternal-ER and protein body-ER), a process that facilitates targeting of their proteins to different endomembrane compartments. Although the cis- and trans-factors responsible for mRNA localization have been defined over the years, how these mRNAs are transported to the cortical ER has yet to be resolved. Here, we show that the two interacting glutelin zipcode RNA binding proteins (RBPs), RBP-P and RBP-L, form a quaternary complex with the membrane fusion factors n-ethylmaleimide-sensitive factor (NSF) and the small GTPase Rab5a, enabling mRNA transport on endosomes. Direct interaction of RBP-L with Rab5a, between NSF and RBP-P, and between NSF and Rab5a, were established. Biochemical and microscopic analyses confirmed the co-localization of these RBPs with NSF on Rab5a-positive endosomes that carry glutelin mRNAs. Analysis of a loss-of-function rab5a mutant showed that glutelin mRNA and the quaternary complex were mis-targeted to the extracellular paramural body structure formed by aborted endosomal trafficking, further confirming the involvement of endosomal trafficking in glutelin mRNA transport. Overall, these findings demonstrate that mRNA localization in plants co-opts membrane trafficking via the acquisition of new functional binding properties between RBPs and two essential membrane trafficking factors, thus defining an endosomal anchoring mechanism in mRNA localization.

RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells.

In developing rice (Oryza sativa) endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.

Developmental and Subcellular Organization of Single-Cell C4 Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization.

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.

RNA targeting to a specific ER sub-domain is required for efficient transport and packaging of α-globulins to the protein storage vacuole in developing rice endosperm.

Studies focusing on the targeting of RNAs that encode rice storage proteins, prolamines and glutelins to specific sub-domains of the endoplasmic reticulum (ER), as well as mis-localization studies of other storage protein RNAs, indicate a close relationship between the ER site of RNA translation and the final site of protein deposition in the endomembrane system in developing rice endosperm. In addition to prolamine and glutelin, rice accumulates smaller amounts of α-globulins, which are deposited together with glutelin in the protein storage vacuole (PSV). In situ RT-PCR analysis revealed that α-globulin RNAs are not distributed to the cisternal ER as expected for a PSV-localized protein, but instead are targeted to the protein body-ER (PB-ER) by a regulated process requiring cis-sorting sequences. Sequence alignments with putative maize δ-zein cis-localization elements identified several candidate regulatory sequences that may be responsible for PB-ER targeting. Immunocytochemical analysis confirmed the presence of α-globulin on the periphery of the prolamine protein bodies and packaging in Golgi-associated dense vesicles, as well as deposition and storage within peripheral regions of the PSV. Mis-targeting of α-globulin RNAs to the cisternal ER dramatically alters the spatial arrangement of α-globulin and glutelin within the PSV, with the accompanying presence of numerous small α-globulin particles in the cytoplasm. These results indicate that α-globulin RNA targeting to the PB-ER sub-domain is essential for efficient transport of α-globulins to the PSV and its spatial arrangement in the PSV. Such RNA localization prevents potential deleterious protein-protein interactions, in addition to performing a role in protein targeting.

Proteomic analysis of cytoskeleton-associated RNA binding proteins in developing rice seed.

In eukaryotes, RNA binding proteins (RBPs) play an integral role not only in RNA processing within the nucleus, but also in the cytoplasmic events of RNA transport, localization, translation, storage and degradation. While many studies have been done, relatively little is known about RBPs in plants. As part of our continuing efforts to understand cytoplasmic gene expression events in developing rice seed (Oryza sativa L.), a proteomics approach was used to identify cytoplasmic-localized, cytoskeletal-associated RBPs. The nucleic acid binding fraction from a cytoskeletal-enriched rice seed extract was isolated by Poly(U)-Sepharose affinity chromatography and analyzed using 2D gel electrophoresis. Analysis of 162 excised protein spots using mass spectrometry led to the identification of 148 distinct proteins, in addition to the highly abundant globulin and glutelin seed storage proteins. Identified proteins include those involved in RNA processing, translation, protein modification, cell signaling, and metabolism, as well as a number of hypothetical proteins. Proteins of particular interest with roles in RNA metabolism are discussed. These results have been deposited within the Rice RNA Binding Protein Database as part of an integrated study of plant cytoskeletal-associated RBPs using developing rice seed as a model.

RiceRBP: A Resource for Experimentally Identified RNA Binding Proteins in Oryza sativa.

RNA binding proteins (RBPs) play an important role not only in nuclear gene expression, but also in cytosolic events, including RNA transport, localization, translation, and stability. Although over 200 RBPs are predicted from the Arabidopsis genome alone, relatively little is known about these proteins in plants as many exhibit no homology to known RBPs in other eukaryotes. Furthermore, RBPs likely have low expression levels making them difficult to identify and study. As part of our continuing efforts to understand plant cytosolic gene expression and the factors involved, we employed a combination of affinity chromatography and proteomic techniques to enrich for low abundance RBPs in developing rice seed. Our results have been compiled into RiceRBP (http://www.bioinformatics2.wsu.edu/RiceRBP), a database that contains 257 experimentally identified proteins, many of which have not previously been predicted to be RBPs. For each of the identified proteins, RiceRBP provides information on transcript and protein sequence, predicted protein domains, details of the experimental identification, and whether antibodies have been generated for public use. In addition, tools are available to analyze expression patterns for the identified genes, view phylogentic relationships and search for orthologous proteins. RiceRBP is a valuable tool for the community in the study of plant RBPs.

RiceRBP: a database of experimentally identified RNA-binding proteins in Oryza sativa L.

RNA-binding proteins play critical roles at multiple steps during gene expression, including mRNA transport and translation. mRNA transport is particularly important in rice (Oryza sativa L.) in order to ensure the proper localization of the prolamine and glutelin seed storage proteins. However, relatively little information is available about RNA-binding proteins that have been isolated or characterized in plants. The RiceRBP database is a novel resource for the analysis of RNA-binding proteins in rice. RiceRBP contains 257 experimentally identified RNA-binding proteins, which are derived from at least 221 distinct rice genes. Many of the identified proteins catalogued in RiceRBP had not previously been annotated or predicted to bind RNA. RiceRBP provides tools to facilitate the analysis of the identified RNA-binding proteins, including information about predicted protein domains, phylogenetic relationships, and expression patterns of the identified genes. Importantly, RiceRBP also contains tools to search and analyze predicted RNA-binding protein orthologs in other plant species. We anticipate that the data and analysis tools provided by RiceRBP should facilitate the study of plant RNA-binding proteins. RiceRBP is available at http://www.bioinformatics2.wsu.edu/RiceRBP.