The N-terminal extension of Arabidopsis ARGONAUTE 1 is essential for microRNA activities

Author summaryGene expression regulation through microRNAs (miRNAs) is widely adopted in eukaryotes. miRNAs are short RNAs ranging from 19 to 24 nucleotides, and associate with a protein named ARGONAUTE (AGO) to form miRNA-induced silencing complexes (miRISCs). A miRISC then binds to a target messenger RNA (mRNA) with sequence complementary to the miRNA, resulting in reduced expression of the target gene. AGO proteins contain four highly conserved domains: N, PAZ, MID and PIWI. Many AGO proteins also possess an N-terminal extension (NTE) region of varying lengths and sequences, of which the function is little known. In plants, most miRNAs are preferentially loaded into AGO1. In this study, we investigated the role of Arabidopsis AGO1 NTE, which is 189 amino acids (a.a.) long and highly unstructured with undefined function. By examining AGO1 mutants with truncated NTE, we show that the 91-to-189a.a. region of the NTE is essential for rescuing the developmental phenotype of an ago1 null mutant and is required for loading miRNAs into AGO1. Moreover, we show that 1-to-90a.a. and 91-to-189a.a. regions of the NTE redundantly promote the activities of AGO1 in the biogenesis of trans-acting small interfering RNAs, a type of endogenous small RNAs that require miRNAs for their biogenesis. microRNAs (miRNAs) regulate target gene expression through their ARGONAUTE (AGO) effector protein, mainly AGO1 in Arabidopsis thaliana. In addition to the highly conserved N, PAZ, MID and PIWI domains with known roles in RNA silencing, AGO1 contains a long, unstructured N-terminal extension (NTE) of little-known function. Here, we show that the NTE is indispensable for the functions of Arabidopsis AGO1, as a lack of the NTE leads to seedling lethality. Within the NTE, the region containing amino acids (a.a.) 91 to 189 is essential for rescuing an ago1 null mutant. Through global analyses of small RNAs, AGO1-associated small RNAs, and miRNA target gene expression, we show that the region containing a.a. 91-189 is required for the loading of miRNAs into AGO1. Moreover, we show that reduced nuclear partitioning of AGO1 did not affect its profiles of miRNA and ta-siRNA association. Furthermore, we show that the 1-to-90a.a. and 91-to-189a.a. regions of the NTE redundantly promote the activities of AGO1 in the biogenesis of trans-acting siRNAs. Together, we report novel roles of the NTE of Arabidopsis AGO1.