Differentiation among the ARGONAUTES

Noncoding RNAs, including small RNAs (sRNAs), act in an astonishing variety of cellular processes, including gene expression, heterochromatin dynamics, and regulation of development (reviewed in Amaral et al., 2008), and ARGONAUTE (AGO) proteins are key effectors of these processes. The Arabidopsis AGOs related to AGO4 are involved in RNA-directed DNA methylation as part of genomic defense against transposable elements (reviewed in Malone and Hannon, 2009). This essential function tames invading transposons by epigenetic suppression of transposon gene expression, thus inhibiting transposition to reduce genomic damage from element movement.

AGO proteins directly bind sRNAs, and different AGOs bind different sRNAs of specific length and preferred 5′ nucleotide. One outstanding question in the field is how much of AGO functional differentiation can be explained by sRNA binding preference and what other factors specify functional differences. Havecker et al. (pages 321–334) examine this question by characterizing the closely related AGO4, AGO6, and AGO9. They first examined the sRNA populations associated with these three AGOs using immunoprecipitation followed by high-throughput sequencing, finding that all three AGOs associated with sRNAs of 24 nucleotides, most with a 5′ adenosine (see figure). However, mapping the associated sRNAs onto the genome showed that the three AGOs showed distinct differences with respect to loci targeted by the sRNAs.

The 5′ nucleotide of sRNAs associated with AGO proteins. Immunoprecipitation followed by high-throughput sequencing was used to characterize the sRNAs associated with AGO4, AGO6, and AGO9. (Figure reproduced from Havecker et al. [2010].)

To determine whether differences in sRNA binding could be attributed to intrinsic properties of the AGO protein or to differences in tissue specificity of AGO expression, the authors made constructs swapping AGO promoters. The sRNA profile of AGO6 or AGO9 expressed from the AGO4 promoter more closely resembled the profile of AGO4, indicating that the AGO expression pattern is important in determining which sRNAs it binds. Examination of AGO promoter-marker gene fusions showed that these three AGOs are indeed expressed in different cell types in embryos and whole plants. However, coincident expression does not explain the full difference: the sRNA profiles from the promoter swap constructs were similar but not identical. This indicates that other factors also determine sRNA binding; moreover, the promoter swap constructs only partially complemented the ago4 mutant phenotype. Indeed, the three AGO proteins showed differences in other biochemical functions, including protein binding and RNA slicing.

This work shows that closely related members of the AGO4 family differ in both their tissue-specific expression and their biochemical properties, leading to functional differences, as shown by the population of sRNAs bound by each AGO. Thus, these closely related members of the AGO family act nonredundantly in genomic defense.