Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2

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Abstract
The association of small RNAs, such as microRNAs (miRNAs), with Argonaute (AGO) family proteins yields complexes that regulate gene expression. While bacterial and archaeal miRNAs show no sequence preference at their 5′ ends, eukaryotic miRNAs tend to have 5′ U or A. To determine the basis of this preference, Bhushan Nagar and colleagues have solved the structure of the human AGO2 MID domain complexed with ribonucleotide monophosphates. The structure reveals a specific interaction of UMP and AMP with a loop that also discriminates against CMP or GMP. The association of microRNAs with Argonaute proteins (AGOs) yields complexes regulating gene expression. Although bacterial and archaeal miRNAs show no sequence preference at their 5′ ends, eukaryotic miRNAs tend to have a 5′ U or A. Here the structure of the human AGO2 MID domain complexed with ribonucleotide monophosphates is solved, revealing specific interaction of UMP and AMP with a loop that discriminates against CMP or GMP, and explaining the observed preference. MicroRNAs (miRNAs) mediate post-transcriptional gene regulation through association with Argonaute proteins (AGOs)1. Crystal structures of archaeal and bacterial homologues of AGOs have shown that the MID (middle) domain mediates the interaction with the phosphorylated 5′ end of the miRNA guide strand and this interaction is thought to be independent of the identity of the 5′ nucleotide in these systems2,3. However, analysis of the known sequences of eukaryotic miRNAs and co-immunoprecipitation experiments indicate that there is a clear bias for U or A at the 5′ position4,5,6,7. Here we report the crystal structure of a MID domain from a eukaryotic AGO protein, human AGO2. The structure, in complex with nucleoside monophosphates (AMP, CMP, GMP, and UMP) mimicking the 5′ end of miRNAs, shows that there are specific contacts made between the base of UMP or AMP and a rigid loop in the MID domain. Notably, the structure of the loop discriminates against CMP and GMP and dissociation constants calculated from NMR titration experiments confirm these results, showing that AMP (0.26 mM) and UMP (0.12 mM) bind with up to 30-fold higher affinity than either CMP (3.6 mM) or GMP (3.3 mM). This study provides structural evidence for nucleotide-specific interactions in the MID domain of eukaryotic AGO proteins and explains the observed preference for U or A at the 5′ end of miRNAs.