Progress 08/01/03 to 07/31/07
Outputs
The Tudor-SN (previously called Rp120) is a major RNA binding activity found in an enriched cytoskeletal fraction. We have previously demonstrated that this RNA binding protein was present in multiple molecular weight, ribonucleoprotein complexes in the cytoplasm, was associated with polysomes, co-localized with GFP-tagged RNA containing particles, and was present as moving particles in live endosperm sections. Moreover, analysis of antisense transgenic plants showed a relationship between Tudor-SN and prolamine protein and RNA levels. Because of the genetic instability of the antisense plants, RNAi plants for Tudor-SN were generated and analyzed. Results showed that reduced gene expression lowered plant growth rates and seed weights. Immunoblot and RT-PCR analysis showed that the RNAi plants had reduced expression of prolamine RNAs and protein while having no effect on expression of protein disulfide isomerase, ubiquitin, and hygromycin acetyltransferase. RNAi plants
also showed lower levels of glutelin polypeptides. Consistent with a role of Tudor-SN in prolamine and glutelin gene expression, immunoprecipitation coupled RT-PCR studies showed that Tudor-SN was bound to prolamine and glutelin RNAs. Microarray analysis is currently being undertaken to identify other Tudor-SN bound RNAs. Collectively, these results corroborate our earlier conclusion that this RNA binding protein is involved in RNA transport and localization to the cortical ER. A manuscript describing these results has been submitted to The Plant Cell for publication. Rice plants expressing TAP antigens fused to intact Tudor-SN and specific Tudor-SN subdomains have been identified and propagated for biochemical analysis. Starting with 200 g of developing seeds, proteins purified by calmodulin and IgG column chromatography have been obtained. Proteins that interact with Tudor-SN are currently being identified using mass spectrometry.
Impacts
RNA localization is a common phenomenon in animal cells and is readily observed in cells with a polarized structure. To date, the only documented case of RNA targeting in plants are the rice storage protein RNAs which are targeted to distinct subdomains of the cortical endoplasmic reticulum in developing rice seeds. The goals of this project are to identify the role of the RNA binding protein Tudor-SN in rice storage protein RNA transport and localization. Information obtained from this study should lead to greater insights on how protein synthesis is controlled in plant cells and may lead to practical applications in improving seed protein quality and quantity and in using developing seeds for the production of value-added proteins.
Publications
- Kumamaru, T., M.Ogawa, H.Satoh, and T.W.Okita. 2007. Protein body biogenesis in cereal endosperms. Endosperm - Developmental and Molecular Biology. Ed. O.-A. Olsen. Springer. 141-158.
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Progress 01/01/06 to 12/31/06
Outputs
The Tudor-SN (previously called Rp120) is a major RNA binding activity found in an enriched cytoskeletal fraction. Results from previous studies showed that this RNA binding protein is present in different molecular species. One form was observed coincident with GFP-tagged RNA particles that have been suggested to mediate RNA transport from the nucleus to the cortical ER. T1 generation transgenic plants expressing Tudor-SN fused to GFP enabled the in situ localization of this RNA binding protein. Tudor-SN was observed as particles located on or close to the prolamine protein bodies. In some instances, speckles of Tudor-SN completely surrounded this organelle. Examination of live endosperm sections showed that several of these Tudor-SN particles move at a velocity and behavior reminiscent of GFP-tagged RNA transport particles. Movement was inhibited by microfilament inhibitors but not by microtubule inhibitors. Collectively, these results corroborate our earlier
conclusion that this RNA binding protein is involved in RNA transport and localization to the cortical ER. Rice plants expressing TAP antigens fused to intact Tudor-SN and specific Tudor-SN subdomains have been identified and propagated for biochemical analysis. Small scale biochemical fractionation studies have shown that a unique collection of polypeptides co-elutes with TAP-Tudor-SN from both the calmodulin and IgG columns. Preparative biochemical studies to identify the nature of these co-eluting polypeptides are underway. Down-regulating Tudor-SN RNAi plants have been generated and analysis has confirmed the down-regulation of prolamine gene expression seen in antisense plants. The RNAi effect on the expression of other genes is currently being evaluated. Although about 1500 nucleotides of the 5' flanking region of the Rp120 gene was fused to a GUS gene, no reporter gene activity was evident in more than 20 transgenic plants surveyed.
Impacts
RNA localization is a common phenomenon in animal cells and is readily observed in cells with a polarized structure. To date, the only documented case of RNA targeting in plants is that involving the rice storage protein RNAs which are targeted to distinct subdomains of the cortical endoplasmic reticulum in developing rice seeds. The goals of this project are to identify the role of the RNA binding protein Rp120 in rice storage protein RNA transport and localization. Information obtained from this study should lead to greater insights on how protein synthesis is controlled in plant cells and may lead to practical applications in improving seed protein quality and quantity and in using developing seeds for the production of value-added proteins.
Publications
- Washida, H., A.J.Crofts, S.Hamada, and T.W.Okita. 2006. Targeting of RNAs to ER subdomains and its relationship with protein localization.. Plant Cell Monogr. (4) The Plant Endoplasmic Reticulum,. Ed. D.G. Robinson. Heidelberg. Springer-Verlag. 25-43.
- Kumamaru, T., M.Ogawa, H.Satoh, and T.W.Okita. 2006. Protein body biogenesis in cereal endosperms. Plant Cell Monographs: Endosperm - Development and Moelcualr Biology. Ed. O.-D. Olsen. Heidelberg. Springer-Verlag.
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Progress 01/01/05 to 12/31/05
Outputs
Results from previous studies showed that Rp120 was present in high molecular RNase sensitive complexes by biochemical analysis. Analysis of gel filtration fractions by native blue PAGE observations suggested the association of Rp120 with L5 and L11 which together with 5S RNA are part of the ribosome large subunit. Immunoblot analysis of ribosomes-polysomes resolved by sucrose density gradient centrifugation showed that Rp120 co-sedimented with monosomes and polysomes suggesting a possible role in translation. The primary sequence of Rp120 contains several potential phosphorylation sites and an on-going study shows that at a significant portion is covalently modified. Earlier attempts failed to express a yeast TAP-Rp120 in transgenic rice. As these yeast TAP sequences have been suggested to contain cryptic splicing sites, studies were repeated with a plant optimized TAP sequence. Rice plants expressing TAP fused to intact Rp120 and specific Rp120 subdomains have been
identified. Small scale biochemical fractionation studies have shown that Rp120-TAP bind to both the calmodulin and IgG columns. Rice plants expressing a GFP-Rp120 fusion have been obtained and the intracellular location is currently being evaluated. GFP-Rp120 is observed as particles which show movement, suggesting a role of this RNA binding protein in RNA transport. Attempts to study the spatial and temporal gene expression patterns of Rp120 promoter failed. Although about 1500 nucleotides of the 5-flanking region of the Rp120 gene was fused to a GUS gene, no reporter gene activity was evident in more than 20 transgenic plants surveyed.
Impacts
RNA localization is a common phenomenon in animal cells and is readily observed in cells with a polarized structure. To date, the only documented case of RNA targeting in plants is that involving the rice storage protein RNAs which are targeted to distinct subdomains of the cortical endoplasmic reticulum in developing rice seeds. The goals of this project are to identify the role of the RNA binding protein Rp120 in rice storage protein RNA transport and localization. Information obtained from this study should lead to greater insights on how protein synthesis is controlled in plant cells and may lead to practical applications in improving seed protein quality and quantity and in using developing seeds for the production of value-added proteins.
Publications
- Crofts, A.J., Washida, H., Okita, T.W., Ogawa, M., Kumamaru, T. and Satoh, H. 2005 The Role of mRNA and Protein Sorting in Seed Storage Protein Synthesis, Transport and Deposition. Biochem. Cell Biol. 83: 728-737.
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Progress 01/01/04 to 12/31/04
Outputs
Results from previous studies showed that Rp120 exists as two distinct populations in developing rice endosperm when viewed by microscopy. One population is closely associated with microtubules while a second type is observed in GFP-tagged prolamine RNA transport particles. More recent studies have detected a third distinct population present in high molecular RNase sensitive complexes by biochemical analysis. Heparin chromatography followed by gel filtration chromatography resolves Rp120 over a very large size distribution ranging from very large macromolecular complexes (> 106 daltons) to about 230 kD (dimmer form). RNase treatment markedly alters the distribution pattern resulting in only the smaller molecular form. These observations indicate that the large Rp120 complexes are formed by interaction with RNA. To further clarify the nature of these large Rp120 complexes, various gel filtration fractions containing partially purified Rp120 were subjected to blue
native polyacrylamide electrophoresis. Two polypeptides co-electrophoresed with Rp120 which were identified as ribosomal proteins L5 and L11 by mass spectrometry. These two ribosomal proteins interact with 5S RNA as part of the ribosome large subunit, suggesting the possibility that Rp120 interacts with ribosomes. Immunoblot analysis of ribosomes-polysomes resolved by sucrose density gradient centrifugation showed, however, that Rp120 did not co-sedmient with monosomes or polysome. TAP-Rp120 expressing rice plants have been obtained but subsequent analysis showed no expression. As these yeast TAP sequences have been suggested to contain cryptic splicing sites, experiments are being repeated with a plant optimized TAP sequence. Rice plants expressing dominant negative mutants of Rp120 gene are being constructed to further explore the role of this RNA binding protein during rice endosperm development. Rice plants expressing a GFP-Rp120 fusion have been obtained and the intracellular
location studied in the next funding period.
Impacts
RNA localization is a common phenomenon in animal cells and is readily observed in cells with a polarized structure. To date, the only documented case of RNA targeting in plants is that involving the rice storage protein RNAs which are targeted to distinct subdomains of the cortical endoplasmic reticulum in developing rice seeds. The goals of this project are to identify the role of the RNA binding protein Rp120 in rice storage protein RNA transport and localization. Information obtained from this study should lead to greater insights on how protein synthesis is controlled in plant cells and may lead to practical applications in improving seed protein quality and quantity and in using developing seeds for the production of value-added proteins.
Publications
- Crofts, A.J. H. Washida, T.W. Okita, T., Kumamaru and H. Satoh 2004 Targeting of proteins to ER-derived compartments in plants: The importance of RNA localization. Plant Physiol. 136:3414-3419.
- Invited seminar presentation, Plant Molecular Biology and Biotechnology (PMBB) Distinguished Lecture, Ohio State University, April 29-30, 2004.
- Invited symposium presentation, Symposium on "Storage Functions in Plants," Tokyo, Japan, August 6, 2004.
- Invited symposium presentation delivered at Symposium on Plant Developmental Biology, International Symposium on Crop Functional Genomics 2004, Cheju (Jeju), Korea, April 7-9, 2004.
- Minisymposium presentation, Haruhiko Washida, Aya Sugino,Tomoyuki Katsube-Tanaka, Chotipa Sakulsingharoj,Sachiyo Kaneko, Changlin Wang, and Thomas W Okita mRNA targeting to endoplasmic reticulum subdomains regulates protein localization in rice endosperm cells. Plant Biology 2004, Annual Meeting of the American Society of Plant Biology Lake Buena Vista, FL.
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Progress 01/01/03 to 12/31/03
Outputs
This project is a continuation of Project No.WNP03589 which is investigating the role of the RNA binding protein Rp120 during endosperm development. Results from previous studies showed that Rp120 exists as two distinct populations in developing rice endosperm. One population is closely associated with microtubules while a second type is found in GFP-tagged prolamine RNA transport particles. To identify proteins that interact with Rp120, immuno-pulldown experiments were conducted. Immunoprecipitation of Rp120 resulted in the selective co-precipitation of at least two other specific polypeptides. Mass spectrometry analysis of generated tryptic peptide fragments of one putative interacting protein identified a protein kinase. Experiments to confirm this initial tentative assignment are currently underway. TAP-Rp120 expressing rice plants have been obtained and are currently being analyzed for potential interacting protein partners. Rice plants expressing dominant
negative mutants of Rp120 gene are being constructed to further explore the role of this RNA binding protein during rice endosperm development.
Impacts
RNA localization is a common phenomenon in animal cells and is readily observed in cells with a polarized structure. To date, the only documented case of RNA targeting in plants is that involving the rice storage protein RNAs which are targeted to distinct subdomains of the cortical endoplasmic reticulum in developing rice seeds. The goals of this project are to identify the role of the RNA binding protein Rp120 in rice storage protein RNA transport and localization. Information obtained from this study should lead to greater insights on how protein synthesis is controlled in plant cells and may lead to practical applications in improving seed protein quality and quantity and in using developing seeds for the production of value-added proteins.
Publications
- No publications reported this period
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