Selective sets of mRNAs localize to extracellular paramural bodies in a rice glup6 mutant.

The transport of rice glutelin storage proteins to the storage vacuoles requires the Rab5 GTPase and its related guanine nucleotide exchange factor (Rab5-GEF). Loss of function of these membrane vesicular trafficking factors results in the initial secretion of storage proteins and later their partial engulfment by the plasma membrane to form an extracellular paramural body (PMB), an aborted endosome complex. Here, we show that in the rice Rab5-GEF mutant glup6, glutelin RNAs are specifically mislocalized from their normal location on the cisternal endoplasmic reticulum (ER) to the protein body-ER, and are also apparently translocated to the PMBs. We substantiated the association of mRNAs with this aborted endosome complex by RNA-seq of PMBs purified by flow cytometry. Two PMB-associated groups of RNA were readily resolved: those that were specifically enriched in this aborted complex and those that were highly expressed in the cytoplasm. Examination of the PMB-enriched RNAs indicated that they were not a random sampling of the glup6 transcriptome but, instead, encompassed only a few functional mRNA classes. Although specific autophagy is also an alternative mechanism, our results support the view that RNA localization may co-opt membrane vesicular trafficking, and that many RNAs that share function or intracellular location are co-transported in developing rice seeds.

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.

Multiple RNA binding protein complexes interact with the rice prolamine RNA cis-localization zipcode sequences.

RNAs for the storage proteins, glutelins and prolamines, contain zipcode sequences, which target them to specific subdomains of the cortical endoplasmic reticulum in developing rice (Oryza sativa) seeds. Fifteen RNA binding proteins (RBPs) specifically bind to the prolamine zipcode sequences and are likely to play an important role in the transport and localization of this storage protein RNA. To understand the underlying basis for the binding of multiple protein species to the prolamine zipcode sequences, the relationship of five of these RBPs, RBP-A, RBP-I, RBP-J, RBP-K, and RBP-Q, were studied. These five RBPs, which belong to the heterogeneous nuclear ribonucleoprotein class, bind specifically to the 5' coding regions as well as to the 3' untranslated region zipcode RNAs but not to a control RNA sequence. Coimmunoprecipitation-immunoblot analyses in the presence or absence of ribonuclease showed that these five RBPs are assembled into three multiprotein complexes to form at least two zipcode RNA-protein assemblies. One cytoplasmic-localized zipcode assembly contained two multiprotein complexes sharing a common core consisting of RBP-J and RBP-K and either RBP-A (A-J-K) or RBP-I (I-J-K). A second zipcode assembly of possibly nuclear origin consists of a multiprotein complex containing RBP-Q and modified forms of the other protein complexes. These results suggest that prolamine RNA transport is initiated in the nucleus to form a zipcode-protein assembly, which is remodeled in the cytoplasm to target the RNA to its proper location on the cortical endoplasmic reticulum.

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.

The small GTPase Rab5a is essential for intracellular transport of proglutelin from the Golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as ß-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.

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.

Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm.

The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides.

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.

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.

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization.

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.

The role of mRNA and protein sorting in seed storage protein synthesis, transport, and deposition.

Rice synthesizes and accumulates high levels of 2 distinct classes of seed storage proteins and sorts them to separate intracellular compartments, making it an ideal model system for studying the mechanisms of storage protein synthesis, transport, and deposition. In rice, RNA localization dictates the initial site of storage protein synthesis on specific subdomains of the cortical endoplasmic reticulum (ER), and there is a direct relation between the RNA localization site and the final destination of the encoded protein within the endomembrane system. Current data support the existence of 3 parallel RNA localization pathways leading from the nucleus to the actively synthesizing cortical ER. Additional pathways may exist for the synthesis of cytoplasmic and nuclear-encoded proteins targeted to organelles, the latter located in a stratified arrangement in developing endosperm cells. The study of rice mutants, which accumulate unprocessed glutelin precursors, indicates that these multiple pathways prevent nonproductive interactions between different classes of storage proteins that would otherwise disrupt protein sorting. Indeed, it appears that the prevention of disruptive interactions between different classes of storage proteins plays a key role in their biosynthesis in rice. In addition to highlighting the unique features of the plant endomembrane system and describing the relation between RNA and protein localization, this minireview will attempt to address a number of questions raised by recent studies on these processes.