Summary Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1–4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad combined with a mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2, without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.

Summary paragraph The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs1–4. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5, glucose metabolism6 and tissue regeneration7. Alternatively, Lin28 overexpression is correlated with the onset of numerous cancers8, while let-7, a tumor suppressor, silences several human oncogenes5. Lin28 binds to precursor let-7 (pre-let-7) hairpins9, triggering the 3' oligo-uridylation activity of TUT4/710–12. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3L213,14, a homolog of the catalytic subunit of the RNA exosome. The molecular basis of Lin28 mediated recruitment of TUT4/7 to pre-let-7 and its subsequent degradation by Dis3L2 is largely unknown. To examine the mechanism of Dis3L2 substrate recognition we determined the structure of mouse Dis3L2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first twelve nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3L2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28/let-7 pathway.

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.

Summary PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.