The expression of genes in eukaryotes is regulated at multiple layers. Transcriptional regulation and post-transcriptional regulation affect the mRNA levels of genes prior to protein translation. In my thesis work, I have worked on two research projects to study gene silencing at transcriptional and post-transcriptional levels in Arabidopsis. First, through a reverse genetic approach, I identified a nucleotidyl transferase HESO1 that functions in the degradation of microRNAs in the Arabidopsis hen1 mutant. As important regulators of gene expression, the accumulation of miRNAs affects the levels of their target mRNAs. I found that a mutation in HESO1 partially rescued the hen1 developmental defects, which result from decreased miRNA levels. The tailing of unmethylated miRNAs in hen1 by HESO1 promoted their degradation by an unknown nuclease, which differed from the one known to cause miRNA 3'-end truncation in hen1. I also confirmed the enzymatic activity of the HESO1 protein in vitro and found that 2'-O-methylation at the 3'-end of small RNAs completely blocked the activity of HESO1. These findings unveiled the protein implicated in small RNA tailing in hen1 and contributed to the knowledge of miRNAs degradation in Arabidopsis. Second, from forward genetic screens using two luciferase reporter lines that are regulated by DNA methylation, I isolated mutants with decreased luminescence and cloned three genes encoding subunits of the THO complex -- TEX1, HPR1 and THO5A. I found that DNA methylation was not affected in the mutants at either the LUCIFERASE transgene locus or most endogenous loci known to harbor DNA methylation. From mRNA sequencing, I identified genes that were up-regulated or down-regulated in tex1 or hpr1. I found that genes associated with dispersed repeats and inverted repeats were enriched among the down-regulated genes in tex1 and hpr1. Additionally, genes with H3K27me3 were enriched in both the up-regulated and the down-regulated genes in tex1 and hpr1. These findings provided insights into the potential targets of THO complex in Arabidopsis.

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.

In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in precursor messenger RNA (pre-mRNA) splicing. In my thesis research, I studied the functions of MAC in RNA metabolism and other aspects of Arabidopsis development and defense. First, we show that the partial loss-of-function mutant mac7-1 and two other MAC subunit mutants, mac3a mac3b and pleiotropic regulatory locus 1 pleiotropic regulatory locus 2 (prl1 prl2), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved eleven known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. mRNA-seq analysis showed that down-regulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants of three subunits of MAC, thus linking MAC function to mRNA splicing in Arabidopsis. Second, since previous studies showed that MAC has multiple functions in eukaryotes, including roles in transcription elongation, mRNA splicing, DNA repair, RNA export and the ubiquitin-proteasome pathway, we explored other potential functions of Arabidopsis MAC and the MAC7 subunit in particular beyond miRNA biogenesis and pre-mRNA splicing. We found that MAC7 has a potential role in photosynthetic pathways, as RNA-seq analysis showed that many photosynthesis-related genes are down-regulated in this mutant. In addition, MAC7 and PRL proteins are very likely to be involved in ribosomal RNA (rRNA) biogenesis, as many rRNA precursors accumulated in the mac7-1 and prl1 prl2 mutants.

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.