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

Abstract

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.

The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana

“…We have also shown that the presence of sDMA modifications is not essential for AtAGO1 miRNA loading and for AtAGO1-dependent SUL silencing triggered by the pSUC2-amiRSUL construct. Our results are consistent with Xu et al who concluded ( 24 ) that the first 90 aa of AtAGO1, which includes 8 out of the 9 RGG/GRG motifs, are not required for miRNA association. In fact, we rather underscore a specific role in the regulation of a subset of 21nt-long siRNAs in prmt5 , mainly originated from genes, and previously identified as phasiRNA ( 57 , 58 , 69 ).…”

“…The N-terminal extensions (NTE) of AGO proteins have been recently analysed in several works ( 17 , 21 , 22 , 24 ) highlighting the main interest on understanding the role of this poorly described region in AGO activity. Our study launches the question of the regulation of AGO1 protein in A. thaliana with a particular emphasis on the role of its NTE, which contains predicted Glycine-Arginine Rich (RGG/GRG) recognition sites for PRMT5 enzyme.…”

“…This region contains NLS and NES sequences which allow AtAGO1 to shuttle between nucleus to load miRNAs, and cytoplasm to regulate corresponding miRNA targets ( 22 ). Moreover, a linear N-coil peptide (F177-K190), located just upstream of the ArgoN domain, is essential for AGO1 dependent miRNA function in A. thaliana ( 17 , 23 , 24 ). Consequently, the presence of RGG/GRG motifs in such an environment prompted us to address the potential role of PRMT5 on AGO1 activity in A. thaliana .…”

Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress

“…Since the Poly-Q domain contains several RGG/RG motifs (Xu et al, 2023) recognized as RNA binding motifs (Thandapani et al, 2013), we then investigated whether the presence of RNA had an influence on LLPS. Interestingly, recombinant GFP- Poly-Q domain AGO1 protein could efficiently bind 112nt long single-stranded and double-stranded RNA in electrophoretic mobility assays (EMSA) (Fig.…”

Interaction between a J-domain co-chaperone and a specific Argonaute protein contributes to microRNA function in animals

“…Strikingly, a recent study in A. thaliana demonstrates that plant AGO1 contains an unstructured N-terminal region (named NTE for N-terminal extension in this study) important for plant viability as well as miRNA stability and loading. (78). This new observation in plants strengthens our ideas that the N-terminal region of AGO is crucial for miRNA loading, and the mechanism we uncovered is likely widespread across different species.…”