RNA silencing mediated by 19-to-24 nucleotide-long small RNAs is a central mechanism regulating gene expression. In plants, the two major types of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are derived from long hairpin RNA precursors while siRNAs are derived from long double-stranded RNAs. In plants, the majority of miRNAs and certain siRNAs associate with the ARGONAUTE1 (AGO1) protein to exert their regulatory effects by forming an RNA-induced silencing complex (RISC). Even though many studies have been done to characterize Arabidopsis AGO1 and AGO1 activities, little is known about the mechanisms that regulate AGO1 RISC formation. In my dissertation research, two mechanisms that regulate AGO1 RISC formation were identified and reported in chapters two and three. In the second chapter, we found that the long structurally unresolved N-terminal extension (NTE) is essential for the functions of AGO1, as an AGO1 mutant lacking this region is unable to rescue the developmental and molecular phenotypes of an ago1 null allele. RNA sequencing of total small RNAs and AGO1-associated small RNAs showed that amino acids (a.a.) 91-189 of the NTE are required for RISC formation, thus crucial for the activities of AGO1 in gene silencing. Furthermore, a.a. 1-90 and a.a. 91-189 regions of the NTE redundantly promote the activities of AGO1 in the biogenesis of trans-acting siRNAs (ta-siRNAs). This work provides a new understanding of the AGO1 NTE region in RISC assembly and ta-siRNA biogenesis. In the third chapter, through immunoprecipitation-mass spectrometry, we found that AGO1 is phosphorylated in vivo. Analysis of AGO1-associated small RNAs in vivo and RISC formation assays in vitro showed that phospho-mimetic AGO1 is compromised in the loading of both miRNA and siRNA duplexes and in passenger strand ejection of siRNA but not miRNA duplexes. Non-phosphorylatable AGO1 is compromised in target RNA repression. This work revealed a regulatory role of phosphorylation and dephosphorylation in the small RNA association and target repression activities of AGO1. Taken together, my dissertation research provided new insights into mechanisms regulating the activities of plant AGO1 and these insights have implications beyond plant AGO1.

microRNAs (miRNAs) play regulatory roles in various developmental processes including stem cell maintenance and differentiation, which are central to animal and plant development. miRNAs function within a miRNA-protein complex termed RNA Induced Silencing Complex (RISC). In plants, miRNAs and the core protein component of RISC, argonaute proteins, have been shown to regulate the maintenance of stem cells. In Arabidopsis, the maintenance of floral stem cell fate is controlled both spatially and temporally. One aim of my Ph.D. thesis research was to uncover the mechanisms that underlie the programmed termination of floral stem cell maintenance. My studies show that miR165/166 and the argonaute protein that it associates with in vivo , AGO10, are both involved in the regulation of floral stem cells. This work is reported in Chapter I of the thesis. One intriguing observation made during my studies was that AGO10 represses the levels of miR165/166 in vivo, which is contrary to the expectation that argonaute proteins stabilize their associated miRNAs and implies the existence of a currently unknown mechanism regulating miRNA homeostasis. My research towards elucidating this regulatory mechanism is reported in Chapter II. Although the work has not been completed, I have accumulated evidence that AGO10 decreases the stability of miR165/166. Taken together, my thesis research has demonstrated that regulation of miRNA stability through a specialized argonaute protein is critical to the regulation of stem cells in plant development.

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.

Although transposons constitute large portions of eukaryotic genomes, certain mechanisms have evolved to suppress the detrimental effects caused by the movement of transposons. In Arabidopsis, DNA methylation plays a vital role in suppressing transposon expression at the transcriptional level, and the underlying mechanisms have been thoroughly investigated. Numerous factors participating in the DNA methylation pathway have been reported, from the establishment of DNA methylation through RNA-directed DNA methylation (RdDM) to the maintenance of symmetrical DNA methylation by MET1/CMT3 and asymmetrical DNA methylation by RdDM and CMT2.

Despite this well-established framework, however, two important questions remain. The first concerns the mystery precursors to siRNAs that function as guidance signals for RdDM. Although it has been proposed that Pol IV transcribes methylated DNA to produce primary transcripts at RdDM loci and that RDR2 converts these transcripts to dsRNAs to serve as siRNA precursors, no such siRNA precursor transcripts have been reported. In my Ph.D. studies, I was able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci through genome-wide profiling of RNAs in genotypes with compromised siRNA precursor processing. On the one hand, Pol IV/RDR2-dependent transcripts differ from Pol II-dependent transcripts in the following ways: they correspond to both DNA strands instead of one strand, they have a 5' monophosphate instead of a 5' cap, they lack a polyA tail at the 3' end, and they do not have introns. On the other hand, both Pol IV/RDR2-transcribed regions and Pol II-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes. Computational analysis of siRNA abundance in various mutants also revealed differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation through two different DNA methyltransferases.

The second question is how the silencing effect of DNA methylation is controlled to prevent the stochastic silencing of genes or to allow the expression of genes that reside nearby transposons. In my Ph.D. studies, I identified SUVH1 as an anti-silencing factor through a forward genetic screen and showed that it promotes the expression of two transgenes and several endogenous genes. 5-Aza-2'-deoxycitidine (a DNA methylation inhibitor) treatment and methylation level analysis using McrBC-PCR and MethylC-seq subsequently showed that SUVH1 functions downstream of DNA methylation to promote the expression of genes harboring promoter DNA methylation. In addition, SUVH1 was found to maintain H3K4me3 levels. These findings from the functional studies of SUVH1 shed light on the regulatory network acting at genes with various epigenetic marks.

Forward genetics is a powerful tool to identify genes involved in particular biological processes. In my thesis work, I participated in forward genetic screens to identify genes involved in two biological processes in plants, stem cell maintenance in the floral meristem and small RNA biogenesis/function. First, I characterized a Polycomb (PcG) gene, CURLY LEAF (CLF) as a factor required for floral meristem termination. A mutation in CLF enhances the floral determinacy defects of ag-10, a weak allele in AGAMOUS (AG), a gene essential for floral stem cell termination. CLF acts in the AG pathway to repress the stem cell identity gene, WUSCHEL (WUS) to result in floral stem cell termination. In addition, I show that this role of CLF reflects the role of the PcG complex in the control of floral meristem determinacy. Taken together, I provide a link between epigenetic regulation and stem cell maintenance in the floral meristem. Second, I identified two genes, MED20a and STERILE APETALA (SAP) that promote small RNA biogenesis and modulate small RNA activities, respectively. MED20a is a subunit of the Mediator complex, which is thought to bridge transcription factors and the RNA Polymerase II (Pol II) transcriptional machinery. In med20a and mutants in two other Mediator subunits, med17 and med18, miRNA accumulation is generally reduced. In the med20a mutant, the levels of pri-miRNA are also lower and the promoter activity of MIR167a is reduced, suggesting that the Mediator promotes MIR gene expression at the transcriptional level. I show that SAP is a negative regulator of the activities of a subset of miRNAs. In sap mutants, the mRNA and protein levels of several miRNA target genes are reduced despite no changes in miRNA accumulation. In addition, some miRNAs are more active in sap mutants, resulting in more 3' cleavage products from target mRNAs. This indicates that SAP negatively regulates the activities of a subset of miRNAs at the posttranscriptional level. These studies extend our understanding of the dynamic control of small RNA biogenesis and function.