Abstracts Category : Other

Contributions of double-stranded RNA binding 2 to the Arabidopsis sRNA landscape

by Kate Chamberlain

Research Doctorate - Doctor of Philosophy (PhD) Small RNA (sRNA)-directed RNA silencing is an essential, conserved mechanism of regulating gene expression in multicellular organisms. <i>Arabidopsis thaliana (Arabidopsis)</i> encodes five DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1 to DRB5, which are collectively required for the establishment and maintenance of the sRNA landscape. In the nucleus, DRB1 is the established cofactor of DCL1 for microRNA (miRNA) production from imperfectly double-stranded RNA (dsRNA) precursor transcripts. Similarly, DRB4 is required by DCL4 for the production of species of small-interfering RNA (siRNA) from endogenous or exogenous derived perfectly dsRNA precursors. Despite considerable investigation into the role of DRB2 in RNA silencing, the precise function of this DRB family member remains ambiguous; DRB2 has been implicated in multiple aspects of RNA silencing, including miRNA biogenesis, translation inhibition of miRNA target genes, and the regulation of 24-nt siRNA production. Therefore, the overarching aim of this Thesis was to further characterise the biological function of DRB2 in the parallel sRNA-directed RNA silencing pathways of <i>Arabidopsis</i>. More specifically, the contribution of DRB1, DRB2, and DRB4 to sRNA production, and thereby transcriptional and post-transcriptional RNA silencing, was compared in three experiments. Firstly, the effect of <i>drb</i> mutation on phosphate (PO) homeostasis was assessed. The PO4 homeostasis pathway was chosen for analysis as the role of miRNAs in mediating phosphate response has been extensively characterised, which provided an opportunity to compare the effects of DRB1, DRB2, and DRB4 on production of these phosphate responsive miRNAs. In plants, miRNA-directed RNA silencing is central in mediating the response to low inorganic phosphate (Pi) availability, and therefore, it was hypothesised that loss of either DRB1, DRB2 or DRB4 would influence the abundance of PO-responsive miRNAs, or the post-transcriptional expression regulation of PO-response genes. It was discovered that DRB1 and DRB2 are integral to PO homeostasis in <i>Arabidopsis</i>. However, unlike DRB1, DRB2 does not participate in the production of PO-responsive miRNAs. Secondly, the potential association between DRB2 and ARGONAUTE7 (AGO7) in the floral <i>TAS3</i>-derived <i>trans</i>-acting siRNA (<i>TAS3</i>-tasiRNA) pathway was assessed. The <i>drb2ago7</i> double mutant plant line, generated in this study for the first time, displayed severe reproductive defects, including increased stigma exsertion, reduced seed set, and shortened siliques. These phenotypic alterations strongly indicated a role for DRB2 in the <i>TAS3</i>-tasiRNA pathway. However, it was determined that DRB2 does not participate in the production of the <i>TAS3</i>-tasiRNA trigger miRNA, miR390, nor does the <i>drb2</i> mutation result in altered accumulation of the tasiARF tasiRNA. Rather, the severe defects expressed by the <i>drb2ago7</i> double mutant are most likely aAdvisors/Committee Members: University of Newcastle. Faculty of Science, School of Environmental and Life Sciences.