Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing

Abstract

Argonaute (AGO) proteins recruit 21-24-nucleotide (nt) small RNAs (sRNAs) to constitute RNA-induced silencing complexes (RISCs) to regulate gene expression at transcriptional or posttranscriptional levels(1-3). Arabidopsis encodes nine functional AGO proteins. These proteins are classified into three clusters, AGO1/5/10, AGO2/3/7 and AGO4/6/9, based on their sequence similarity, functional redundancy, as well as species and features of AGO-bound sRNAs(4-7). Although most Arabidopsis AGO proteins have been studied well, AGO3-bound sRNAs and their basic function remain unknown. Here we observed that AGO3 could not complement the signature function of AGO2, the closest genetic paralog of AGO3, in host antiviral defence. We also found, surprisingly, that AGO3 predominantly bound 24-nt sRNAs with 5'-terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2, further indicating their functional divergence. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4, and over 60% of AGO3-associated 24-nt sRNA-enriched loci were identical to those of AGO4. Moreover, the redundancy of AGO3- and AGO4-bound sRNAs is much more than that of AGO6- and AGO4-recruited sRNAs. In addition, expression of AGO3 driven by the AGO4 promoter partially complemented AGO4 function and rescued a DNA methylation defect in the ago4-1 background. Together, our results indicated that AGO3, similarly to AGO4, is a component in the epigenetic pathway.

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

“…At the same time, however, several genes in this region of chromosome I display polymorphisms including AGO3 . Phylogenetically, AGO3 is the closest homolog to AGO2 , yet except against BaMV, AGO3 has not been found to be involved in virus resistance (Jaubert et al , ; Brosseau & Moffett, ; Garcia‐Ruiz et al , ; Brosseau et al , ; Alazem et al , ), but rather appears to play a role in DNA methylation (Zhang et al , ). Moreover, although AGO3 exhibits some polymorphisms, in silico analysis revealed no correlation between any of these polymorphisms and PVX susceptibility (Table S5b).…”

Dynamic changes of small RNAs in rice spikelet development reveal specialized reproductive phasiRNA pathways

“…Our data showed that OsAGO18 and OsAGO2b transcripts increase substantially at stage 7, coincident with 24-nt phasiRNA accumulation. Moreover, a recent study on Arabidopsis AGO3 showed that this poorly characterized Argonaute mediates RdDM by binding 24-nt siRNAs (Zhang et al , 2016). Phylogenetic analysis showed that Arabidopsis AGO3 and rice OsAGO2b are close to each other (Zhang et al , 2015), suggesting that OsAGO2b could also recruit 24-nt small RNAs, such as the reproductive phasiRNAs, to regulate epigenetic modifications.…”

Small RNA-Sequencing Links Physiological Changes and RdDM Process to Vegetative-to-Floral Transition in Apple

“…Six micro-grams (μg) of total RNA were spiked with 5′ 32 P-labeled radioisotope-labeled 19–24 nt RNA oligos and then resolved in 15% urea-PAGE. sRNA library was constructed as previously described (Zhang et al, 2016). Briefly, sRNAs of 19–24 nt were gel-purified and ligated to a pair of adapters at the 5′ and 3′ ends by using T4 RNA ligase.…”

“…sRNA blot assays were performed with aliquots of total RNAs used for sRNA-seq according to Zhang et al (2016). Each lane contained 6 μg of total RNA.…”