Allosteric regulation of Argonaute proteins by miRNAs

Abstract

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) bind to Argonaute family proteins to form a related set of effector complexes that play diverse roles in post-transcriptional gene regulation throughout the eukaryotic lineage. Here, sequence and structural analysis of the MID domain of the Argonaute proteins identified similarities with a family of allosterically regulated bacterial ligandbinding domains. In vitro and in vivo approaches were used to show that certain Argonaute proteins (those involved in translational repression) have conserved this functional allostery between two distinct sites, one involved in binding miRNA:target duplex and the other in binding the 5' cap feature (m 7 GpppG) of eukaryotic mRNAs. This allostery provides an explanation for how miRNA-bound effector complexes may avoid indiscriminate repressive action (mediated through binding interactions with the cap) prior to full target recognition. KeywordsArgonaute; miRNA; miRNP; m 7 GpppG cap; allostery Small interfering RNAs (siRNAs) and microRNAs (miRNAs) belong to an increasingly broad class of small, non-coding RNA molecules found in diverse organisms 1 . These two groups are broadly distinguished by their biogenesis pathways and their differential loading into distinct Argonaute complexes. siRNAs are generated in the cytoplasm and are loaded into an Argonaute-containing RNA-induced silencing complex (siRISC) to cleave targets with perfect complementarity. miRNAs are transcribed in the nucleus from a specific gene and are ultimately loaded into Argonaute-containing miRNPs (miRISC) that limit the expression of distinct mRNA targets with imperfect complementarity. And, though there are clearly shared molecular features for these two processes (e.g. an Argonaute protein and a small RNA), there are also distinct molecular features and players important both for small RNA loading and target recognition 2 . The Argonaute proteins are composed of several distinct domains with partially understood functions: an N-terminal domain, a PAZ domain that contains the binding site for the 3' end of the small RNA, a MID domain that contains the binding site for the 5' end of the small RNA (which will be the focus of discussion in this manuscript), and the PIWI domain that contains the catalytic center for the cleavage reaction that occurs during RNA interference (Fig. 1a).A number of molecular mechanisms have been proposed to account for the observed posttranscriptional control of miRNA-targeted genes including inhibition of translation initiation or elongation and the promotion of mRNA decay (reviewed in ref. 3). One previous studyCorrespondence should be addressed to R.G. (ragreen@jhmi.edu).. * These authors contributed equally to this work. Author InformationThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2010 March 8. RESULTS Argonaute MID domain analysis reveals functional groupingsThe mechanistic questions that arose from the studies b...

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

“…However, despite these similarities, the PIWI-domain exhibits eukaryote-specific structural features that might act as sensors for the functional state of the protein. Finally, we show that residues that have been implicated in allosteric regulation in previous studies (21) are in fact involved in MID-PIWI interdomain interactions and contribute to guide RNA binding by stabilizing the MID-PIWI interface.…”

“…Relevant side chains and secondary structure elements are labeled. D603 corresponds to the aspartate proposed by Djuranovic et al (21) to mediate allosteric regulation of miRNA binding. MID, pale green; PIWI, yellow.…”

“…16) of the PIWI domain revealed an interesting conformational difference in a loop between residues D603 and V608 (termed loop L D603 ). This loop had previously been implicated in the allosteric regulation of guide RNA binding, and mutational analysis has suggested that an aspartate (D603 in Nc QDE-2) could mediate this effect (21); this residue is conserved in a subset of eukaryotic AGOs (Fig. S1).…”

Regulation of miR106b cluster through the RB pathway

“…MCF7 cells were treated with DMSO or PD 0332991 for 24 h, and the RNA immunoprecipitation was performed using Ago2 antibody as described. 48,49 RNA was extracted from immunoprecipitates using Trizol-Reagent (Invitrogen). cDNA was synthesized, and qRT-PCRs were performed for p21, PTEN mRNA and miR106B using specific microRNA probes as described in manufacturer's protocol (Applied Biosystem).…”

MicroRNA-mediated gene silencing: are we close to a unifying model?

“…Therefore, it remains to be determined whether this observed interaction with the m 7 G-cap structure is mediated via another region of Ago 2 or indirectly by an unidentifi ed intermediary protein. Subsequent studies modelling the mid-domain indicate the presence of a site able to bind nucleotides, such as m 7 G-cap, and that allosteric control of this site dependent on miRNA binding provides specifi city to such interactions (45) .…”