Dissertation
Isolation, identification and characterization of RNA binding proteins involved in prolamine MRNA localization in rice endosperm cells
Washington State University
Doctor of Philosophy (PhD), Washington State University
08/2009
DOI:
https://doi.org/10.7273/000005917
Abstract
RNA localization is an evolutionarily conserved mechanism for targeting gene products to specific subcellular locations. The best-studied example of RNA localization within the plant kingdom is the sorting of the mRNAs encoding the rice seed storage proteins, prolamine and glutelin, to distinct endoplasmic reticulum (ER) subdomains. In developing rice seed endosperm, prolamine mRNAs are highly enriched on ER membranes which bound spherical protein bodies (PB-ER) formed by the controlled aggregation of nascent prolamine polypeptides. In contrast, glutelin mRNAs are enriched on adjacent cisternal ER membranes and, following translation, the encoded glutelin precursors are exported to the protein storage vacuole. RNA localization signals, known as zipcodes, have been identified in both prolamine and glutelin mRNAs and are responsible for their distinct localization patterns. Since it is known that prolamine mRNAs are transported via the actin cytoskeleton in a zipcode-dependent manner, trans-acting RNA binding proteins must be required to localize these mRNAs to the PB-ER. Therefore, RNA binding proteins (RBPs) were isolated from cytoskeleton-enriched seed extracts using a prolamine zipcode affinity column, resulting in the identification of eighteen proteins by mass spectrometry. Affinity purified antibodies were generated against fifteen of these, including three chloroplast RBPs and seven members of the heterogeneous ribonucleoprotein (hnRNP) family which have known roles in RNA targeting. RBP-A, RBP-D and RBP-I, all of which are hnRNPs, show specificity for prolamine zipcode RNA compared to control RNA. The expression profiles of RBP-A and RBP-D show that both proteins are highly expressed only during the most rapid phase of storage protein accumulation. Microscopy data for RBP-A and RBP-D is also consistent with a role in RNA localization. Both are present on ER membranes and in particles which are closely associated with cytoskeletal elements. The development of a novel approach based on RNA immunoprecipitation coupled with microarray analysis allowed the identification of RNA species interacting with RBP-A. These included both prolamine and glutelin, in addition to a number of others. This data therefore supports a role for RBP-A as an RNA binding protein involved in the localization of storage protein RNAs to ER membranes.
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Details
- Title
- Isolation, identification and characterization of RNA binding proteins involved in prolamine MRNA localization in rice endosperm cells
- Creators
- Naoko Crofts
- Contributors
- Thomas W. Okita (Chair)Lee A Hadwiger (Committee Member) - Washington State University, Department of Plant PathologyChulHee Kang (Committee Member) - Washington State University, Department of ChemistryAndrew McCubbin (Committee Member) - Washington State University, School of Biological SciencesB W Poovaiah (Committee Member) - Washington State University, Department of Horticulture
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 151
- Identifiers
- 99901055133201842
- Language
- English
- Resource Type
- Dissertation