Investigating the Mechanism of MicroRNA Turnover and Characterizing the Role of RCD1 During Small RNA Metabolism in Arabidopsis
Small RNAs are a class of non-coding RNAs that mediate gene silencing at both transcriptional and post-transcriptional levels in eukaryotes. In plants, microRNAs (miRNAs) and small interfering RNAs (siRNAs) are two core types of small RNAs, which play essential roles in diverse biological processes. Both the metabolism and activities of small RNAs contribute to the regulation of their targets. Even though many on small RNA pathway have been reported, the detailed mechanism on its metabolism is still lacking. To address this issue, my thesis research has employed multidisciplinary approaches and techniques. The achieved results and conclusions can be closed as the following. First, it was found in this research that an Argonaute protein AGO10 represses the accumulation of miR165/6 without affecting its biogenesis. AGO10 overexpression results in a reduction in full-length miR165/6 species accompanied with an elevated 3' truncation of miR165/6, which is rescued by losses of two exonucleases SDN1 and SDN2. An enzymatic assay between SDN1 and AGO10 immunoprecipitates in vitro was carried out, which confirms that AGO10 represses the accumulation of miR165/6 through promoting its degradation via SDN1. A similar assay between SDN1 and AGO1 immunoprecipitates was also performed, allowing the proposal of a model on miRNA degradation. This is the work reported in the Chapter Two of this dissertation. Second, in this research, through small RNA sequencing and comparison to wild type, many siRNAs were identified to be differentially expressed in the rcd1 mutant (int51). A whole-genome bisulfate-sequencing reveals that DNA methylation in all sequence contexts is greatly reduced on euchromatic arms in int51, while is not changed at heterochromatic regions. It was further found that the global level of H3K9me2 is reduced in int51. The changes observed in int51 resemble those of mutants defective in DNA or histone methylation, providing a novel insightful hint to dissect the RCD1-mediated regulatory network further. My research towards elucidating this network is reported in Chapter Three. Taken together, my dissertation research has established new understanding on the mechanism of plant miRNA degradation and has also identified a novel candidate in plant siRNA metabolism.