The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation

Abstract

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1–10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5′ nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5′ adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5′ A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

The clade-specific target recognition mechanisms of plant RISCs

“…This duplex had mismatches at positions 5, 9, and 16 to enhance passenger strand ejection. It should be noted that although this duplex lacks a mismatch at position 1, the guide strand selection is preferentially biased, as one strand’s 5′ terminal base is A while the other strand’s is G, as described by Liu et al, 2022 (44).…”

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation

“…Previously, studies have been undertaken concerning the structure of the MID domain of Argonaute proteins, highlighting its responsibility for the selective recognition of the 5′ nucleotide (Frank et al 2012 ). Interestingly, a coordination between the MID and PIWI domains has been identified as the underlying reason for this specificity (Liu et al 2022 ). In our study, we found that, among the conserved sites involved in 5′ nucleotide anchoring, only one site differed between AtAGO4, HvAGO4A and HvAGO4B, which was conserved between rice and barley (Fig.…”

“…Indeed, in Arabidopsis , AGO6 was found to be able to load 24-nt sRNAs, as well as shorter sRNAs (19–22 nt), but with an exclusive preference for sRNAs with A residues at their 5′ ends. These length- and nucleotide-specific features ensure non-competition with other AGO proteins, particularly AGO1, which primarily binds miRNAs characterized by a length of 21 nucleotides and a starting base of U (McCue et al 2015 ; Liu et al 2022 ). This diversification might play a more specialized role in barley and tissues where both AGO4 proteins are expressed, potentially involving distinct localizations at the cell type level between the two.…”

“…AGO proteins consist of the following domains: N-terminal; PIWI/Argonaute/Zwille (PAZ); MID; P-element-Induced WImpy testis (PIWI) domains and two linkers (Hutvagner and Simard 2008 ). The PIWI domain forms a pocket with the MID domain that recognizes the 5′-end of the sRNA (Zhang et al 2014a ; Liu et al 2022 ). Based on their length, and the nucleotide at the 5′-end, sRNAs can be sorted into different AGO proteins.…”

Emerging Roles of Epigenetics in Grapevine and Winegrowing

“…Next, Pol IV transcribes these regions producing short single-stranded RNAs (ssRNAs), which are then converted into double-stranded RNAs (dsRNAs) by the RNA-directed RNA polymerase 2 (RDR2) [ 31 ]. These dsRNAs are cleaved by the endoribonuclease enzyme DCL3 into 24-nucleotide small interfering RNAs (siRNAs) and are loaded onto Argonaute 4 (AGO4) or AGO6 proteins forming an AGO–siRNA duplex that enables AGO proteins to recognize and bind to RNA sequences complementary to the siRNA partner [ 32 ]. The second stage of this pathway involves the recruitment of Pol V to chromatin by DNA methyl readers SUVH2 and SUVH9 together with the DDR complex (consisting of DMS3, DRD1, and RDM1), leading to the synthesis of Pol V non-coding transcripts [ 33 , 34 ].…”