Systematic studies on the recognition of 3′‐end methylated small silencing RNAs by PAZ domains

Abstract

The PAZ domain is a conserved RNA‐binding module that is only found in two core components of RNA silencing, Argonaute and Dicer. Previous studies showed that the PAZ domain specifically recognizes 3′‐end 2‐nucleotide overhang of small RNAs. Recently, it has been demonstrated that many small silencing RNAs, such as miRNAs and siRNAs in plants, piRNAs in animals and siRNAs in Drosophila, are methylated on the 2′‐OH of the 3′ terminal nucleotide. This kind of modification resulted in a reduction of binding affinity between the small RNAs and the PAZ domain from human AGO1. In order to further understand the function of the PAZ domain from different Argonaute proteins, systematic binding and structural studies of PAZ domains derived from two subfamilies of Argonaute proteins (AGO and PIWI) and four species (human, fly, fish and worm) are carried out. Results from different binding assays (fluorescence anisotropy and isothermal titration calorimetry) indicate that PAZ domains from human and Drosophila AGO1 proteins bind to 3′‐end methylated small RNAs with reduced binding affinities but PAZ domains from Drosophila AGO2 and other PIWI subfamily Argonaute proteins bind to 3′‐end methylated small RNAs with enhanced binding affinities, which consist with deeper and wider pockets found in PAZ domains from Drosophila AGO2 and PIWI subfamily proteins. Structural comparison implies that PAZ domains from divergent Argonaute proteins may have distinct functions.