Arabidopsis thaliana under stress: Functional implications of G-quadruplex and non-coding RNA structure

Open Access
Mullen, Melissa Ann
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
July 07, 2011
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Chair
  • Sarah Mary Assmann, Committee Chair
  • Christine Dolan Keating, Committee Member
  • David D Boehr, Committee Member
  • Paul Lee Babitzke, Committee Member
  • RNA
  • Arabidopsis
  • G-quadruplex
  • drought stress
RNA can adopt diverse secondary and tertiary structures including duplexes, hairpins, quadruplexes, and pseudoknots. Changes in RNA structures have been shown to affect gene expression in all domains of life. In plant systems survival is dependent on stress responses to changing environmental conditions, including drought stress. Increases in ionic strength, K+, and osmolyte concentrations in response to stress affect RNA folding thus RNA structure that can refold under such conditions has the potential to have a regulatory effect. The objective of this work is to investigate RNA folding in the model plant system Arabidopsis thaliana under normal and stress conditions and to evaluate the potential for RNA-mediated gene regulation using bioinformatics, biochemistry, and biology. G-quadruplex sequences (GQS), which require K+ for formation, have been shown to regulate gene expression in H. sapiens. Using computational methods, I found that GQS of varying motifs are present in Arabidopsis thaliana and that the distribution of GQS varies with sequence motif. Using a Markov modeled genome we determined that GQS with three G-tracts (G3) are significantly underrepresented in the genome. Functional analysis was performed on GQS present in RNA indicated that GQS are overrepresented in genes encoding proteins of certain functional categories, including enzyme activity. I also found that genes differentially regulated by drought were significantly more likely to contain a GQS. Circular dichrosim (CD)-detected K+ titrations and UV thermal denaturation performed on representative RNAs verified that quadruplexes formed at physiological K+ concentrations. Results from this study indicate that GQS are present in unique locations in A. thaliana and that folding of RNA GQS may play important roles in regulating gene expression. In vitro folding of GQS under normal and drought stress conditions was investigated using biochemical techniques. We found that RNA GQS form with folding parameters that depend on loop length, sequence and G-tract length. Folding parameters for RNA GQS were determined using CD-detected K+ titrations and indicated that RNA sequences with G3 had a modest dependence on K+ concentration and folded with negative cooperativity. Native gel analysis and RNase T1 protection assays revealed that G3 GQS are associated with populated intermediate folding states. GQS with two G-tracts (G2) had a steep K+ dependence, folded with a high cooperativity factor, and did not significantly populate intermediate states. In a plant system under unstressed conditions, the more stable G3 sequences were predicted to fold, however the less stable G2 sequences were only predicted to have a favorable folding free energy at high K+ concentrations, which are found during drought stress. GQS in the RNA have the potential to switch structure in response to high K+ conditions and thus regulate gene expression as a drought stress response. Some heavy metals, including Cu2+, Zn2+, and Fe2+ are essential for plant metabolism and normal function; however they are toxic in high concentrations. Heavy metal interactions with DNA GQS were monitored by CD. Results from the analysis indicate that Cu2+ interacts with DNA GQS more strongly than other heavy metal ions and destroys the quadruplex structure. Additionally, RT-PCR results indicated that expression of some copper transport genes that contain a GQS is up-regulated when in plants grown on Cu2+-supplemented media. This response could be a mechanism to survive an excess of heavy metal ions in the local environment. Finally, a study of the structure of the 3’UTR of the abscisic acid (ABA) catabolism gene CYP707A4, was conducted to characterize structure and potential binding interactions with ABA. ABA, a plant hormone that mediates the drought stress response in Arabidopsis, increases in concentration to µM levels under drought stress. The AU-rich 3’UTR of CYP707A4 has an ion-dependent structure and undergoes an RNA refolding event with Mg2+ and K+, as determined using UV-detected thermal denaturation, CD, and hydroxyl radical footprinting. Isothermal titration calorimetry, circular dichroism, and equilibrium dialysis were used to test for ABA binding to the 3’UTR of CYPY707A4. Results from these techniques suggest at most a weak and non-specific interaction with ABA.