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Characterization of Self-Interaction of Arabidopsis thaliana Double-Stranded RNA Binding Protein 4

Singh, Jasleen
http://rave.ohiolink.edu/etdc/view?acc_num=osu1337822686

Abstract Details

2012, Master of Science, Ohio State University, Plant Pathology.
The interaction between Arabidopsis thaliana double stranded RNA binding protein 4 (DRB4) and dicer-like 4 (DCL4) has been shown to be necessary for RNA silencing. In addition, DRB proteins may also facilitate the loading of siRNAs into RISC. However a mechanistic understanding of their role in anti-viral silencing requires a detailed knowledge of interactions between the silencing pathway components, as well as interactions between different domains of the individual components. Here we report our efforts at resolving these issues using both the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Using both BiFC and Y2H assay we show that DRB4 interacts with itself, and that it’s N-terminal half, consisting of two dsRNA binding motifs (dsRBMs), mediates this interaction. Splitting these two dsRBMs resulted in a complete loss of interactions in yeast. Conversely, the C-terminal half of DRB4 was unable to self-interact, and was completely dispensable for DRB4 dimerization. We further delineated the amino acid residues essential for this interaction by mutating the highly conserved amino acid residues within the two dsRBMs. Mutating both the histidine and lysine residues, at position 32 and 133 respectively, led to a complete loss of DRB4 self-interaction in yeast. Interestingly, these amino acids have been previously shown to be critical for the DRB4/DCL4 interaction. Our current data suggests that DRB4 likely exist as a dimer in cells and this dimerization is necessary for its interaction with DCL4 and hence anti-viral RNA silencing. Our findings provide a better understanding of the molecular events at the initiation step of anti-viral RNA silencing pathway in Arabidopsis thaliana.
Feng Qu, Dr. (Advisor)
Thomas Mitchell, Dr. (Committee Member)
Lucy Stewart, Dr. (Committee Member)
103 p.
Biology; Molecular Biology; Plant Biology; Plant Pathology; Plant Sciences
RNA silencing; anti-viral defense; double stranded RNA binding proteins; DRB4

Recommended Citations

Citations

  • Singh, J. (2012). Characterization of Self-Interaction of Arabidopsis thaliana Double-Stranded RNA Binding Protein 4 [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337822686

    APA Style (7th edition)

  • Singh, Jasleen. Characterization of Self-Interaction of Arabidopsis thaliana Double-Stranded RNA Binding Protein 4. 2012. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1337822686.

    MLA Style (8th edition)

  • Singh, Jasleen. "Characterization of Self-Interaction of Arabidopsis thaliana Double-Stranded RNA Binding Protein 4." Master's thesis, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337822686

    Chicago Manual of Style (17th edition)

osu1337822686
867
© 2012, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.