Effects of Sequence Complementarity and Argonaute Slicing Activity on RNA-Dependent DNA Methylation in Arabidopsis

Open Access
Page, Charles A
Area of Honors:
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Michael Axtell, Thesis Supervisor
  • Stephen Wade Schaeffer, Honors Advisor
  • Biology
  • Plant Biology
RNA-directed DNA Methylation (RdDM) is an important regulator of gene expression. In plants, RdDM involves the Argonaute 4 (AGO4) family of proteins, as well as 24 nucleotide (nt) short RNAs. AGO4 is known to have a domain with slicing activity towards bound RNAs, but the significance of this slicing activity is not known. Likewise, previous work has shown that 24nt RNAs guide RdDM based on complementarity with the DNA to be methylated, but the degree of complementarity required is unknown. Here we examine several methods to further investigate these mechanisms. We show that an AGO4 transgene with three slicing catalytic residues altered (AGO4-AAA) does not accumulate sufficiently in vivo to test its efficacy. A 24nt RNA generating transgene based on rice 24nt microRNAs is also found not to accumulate sufficiently in vivo for further use as a system for testing the complementarity requirements of RdDM. A third system, based on inverted repeat RNA (irRNA) is shown to successfully accumulate and induce RdDM at a target locus, opening the irRNA system as a basis to test RdDM complementarity requirements in future experiments.