Identification of Novel Mechanisms that Regulate the Activities of ARGONAUTE1 in Arabidopsis thaliana

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