Multivalent Recruitment of Human Argonaute by GW182

Abstract

Summary In miRNA mediated gene silencing the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2 and the hook motif. The structure of hAgo-1 in complex with the hook motif of hGW182 reveals a “gate”-like interaction that is critical for GW182 docking into one of hAgo1’s tryptophan binding pockets. We show that hAgo-1 and -2 have a single GW182-binding site and that miRNA binding increases hAgo’s affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.

Expression of TNRC6 (GW182) Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes

“…The N-terminal repeat GW-repeat containing region of the TNRC6 proteins binds to argonaute (AGO) protein [9,[11][12][13][14]. The complex between AGO and guide RNA is responsible for sequence-selective recognition, whereas the TNRC6 paralogs recruit interacting factors such as the CCR4-NOT complex to mediate gene expression [15][16][17][18].…”

Direct interaction of PIWI and DEPS-1 is essential for piRNA function and condensate ultrastructure inCaenorhabditis elegans

“…The C-terminal region with similarity to dmGW182 is the poly-serine tail. Intriguingly, the N-terminal region of DEPS-1 similar to dmGW182 shares similarity with dmGW192's Ago-binding motif II ( Figure 1C; Elkayam et al, 2017;Eulalio et al, 2009). Moreover, this N-terminal region is contained within DEPS-1 frag1 which binds to PRG-1 PIWI .…”

“…So far only a few examples of direct interactors for the Ago protein family, in particular the PIWI clade, have been identified. A sub-micromolar dissociation constant is on par with GW182 and Tudor domain-containing proteins interaction with human Ago and mouse PIWI (Elkayam et al, 2017;Zhang et al, 2017) indicating DEPS-1 and PRG-1 binding is physiologically relevant.…”

CRISPR screening strategies for microRNA target identification

“…Examining synthetic effects would be much more technically demanding because (a) depending on library complexity, the number of transduced cells required to sample all pairwise combinations of guides could be exorbitant and (b) extensive cloning and barcoding would be required to determine when more than one sgRNA or mutation is in the same cell, though an adapted single-cell sequencing platform might facilitate this. Nonetheless, examining miRNA binding sites individually may be very powerful because miRNAs often act cooperatively at multiple sites on a single 3 0 UTR [43][44][45][46][47]. In these cases, disruption of a single site would be expected to have a large effect on target repression and might have phenotypic effects despite other sites remaining intact.…”