The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins

Abstract

Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

Identification of Potential microRNAs and Their Targets in Brassica rapa L.

“…It is suggested that the difference in size of the identified miRNAs within different families might offer unique functions for the regulation of miRNA biogenesis or gene expression in plants. It is reported that uracil is more represented at the 5′ side of mature miRNAs; the percentage of representation of mature miRNAs having U at the 5′ side of the stem loop is 66.67%, followed by A (14.52%), C (9.68%), and G (9.14%); this is consistent with previous findings (Mi et al, 2008;Montgomery et al, 2008;Takeda et al, 2008;Zhang et al, 2008). It is speculated that the strong bias of uracil in the first 5′ nucleotide position is due to its important role in the recognition of the miRNA by argonaute1 (Zhang et al, 2006b Table 1 and Fig.…”

Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly

“…In contrast to AGO7, AGO2 efficiently incorporated the miR390/miR390* mutant with the flipped central 3-nt ( Supplementary Fig S1 online), suggesting that AGO2 does not inspect the central region for RISC assembly. This is consistent with the previous finding that AGO2 incorporates a wide variety of 5 0 -A small RNAs including viral siRNAs [22][23][24]. miR390/miR390* duplex has two mismatches: a G-U wobble in the seed region (guide position 3) and a G-A mismatch in the central region (guide position 11).…”

Genome-wide profiling of sRNAs in the Verticillium dahliae-infected Arabidopsis roots

“…sRNAs with length between 20 and 25 nt were included in our analysis. Previous studies reported that Arabidopsis sRNAs are sorted into distinct AGO complexes depending on the length and 5’ first nucleotide of sRNAs, which has important implications for the functions of miRNAs and siRNAs (Mi et al 2008; Takeda et al 2008). Therefore, we detected the length and 5’ first nucleotide of sRNAs in the two libraries respectively.…”