Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression

In mammalian cells, microRNAs (miRNAs) are incorporated into miRNA-induced silencing complexes (miRISCs), which regulate protein expression post-transcriptionally through binding to 3′-untranslated regions of target mRNAs. Argonaute2 (Ago2), a key component of the miRISC, recruits GW182, a component of the processing body (GW/P-body), to the target mRNAs. To elucidate the function of GW182 in an miRNA-mediated translational repression, we analyzed Argonaute-binding sites in GW182. We found that human GW182 contains three binding sites for Ago2, within the amino-terminal glycine tryptophan (GW/WG)-repeated region that is characteristic of the GW182 family proteins. We also found that the first and second Ago2-binding site is conserved within the amino-terminal half of TNRC6B, which is a paralog of GW182. Each of the Ago-binding sites is alone sufficient to bind Ago2. Furthermore, we demonstrated that multiple Argonaute proteins were connected via the GW182 protein. A GW182 fragment containing the Ago2-binding region partially relieved let-7-mediated repression of protein synthesis in a mammalian cell-free system. Coincidentally, let-7-directed target mRNA deadenylation was delayed. Together, these results strongly suggested that the interactions of GW182 with Argonautes may induce the formation of large complexes containing miRNA target mRNAs, and may be critical for miRNA-mediated translational repression

Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system

MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3′-untranslated regions of target mRNAs. Here we describe a recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5′-cap and 3′-poly(A) tail, resulting in translational repression

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain

Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction

<div><p>Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody–antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059–152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355^EGFR, Gln384^EGFR, H409^EGFR, and Lys465^EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.</p></div

SDS-PAGE analysis of site-specific fluorescent-labeled 059-152-Fv and 059-152-Fab.

<p>Reducing and non-reducing SDS-PAGE analysis of 059-152-Fv (A) and 059-152-Fab (B). (lane 1) 059-152-Fv, (lane 2) AzF-incorporated 059-152-Fv, (lane 3) Alexa-488 conjugated 059-152-Fv, (lane 4) 059-152-Fab, (lane 5) AzF-incorporated 059-152-Fab, and (lane 6) Alexa-488 conjugated 059-152-Fab. Fluorescent images and CBB-stained images were acquired from the same gels.</p

SDS-PAGE analysis of cell-free synthesized 059-152-Fv and 059-152-Fab.

<p>(A) 059-152-Fv was synthesized under a series of different concentrations (0, 0.2, and 0.4 mg/ml) of DsbC, as indicated. Total (T) and soluble (S) fractions of the internal solution were analyzed by reducing SDS-PAGE. (B) Purified 059-152-Fv was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fv are indicated under each lane of the non-reducing SDS polyacrylamide gel image. (C) 059-152-Fab was synthesized in the presence of 0, 0.2, 0.4, and 0.8 mg/ml of DsbC, as indicated. (D) Purified 059-152-Fab was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fab are indicated under each lane of the non-reducing SDS polyacrylamide gel image. BG: cell-free synthesis without template DNA. VH: cell-free synthesis of VH without DsbC. VL: cell-free synthesis of VL without DsbC. VHCH1: cell-free synthesis of VHCH1 without DsbC. L chain: cell-free synthesis of light chain without DsbC. Gels were stained with CBB.</p

Crystal structure of the 059-152-Fv•EGFR-ECD complex.

<p>(A) Ribbon representation of the 059-152-Fv•EGFR-ECD complex. VH and VL domains of 059-152-Fv are respectively colored cyan and green. External region of the EGFR is shown with domain I in yellow, domain II in orange, domain III in red, and domain IV in purple. (B, C) Close up-view of the interactions between CDR loops of 059–152 and domain III. For clarity, interactions of CDR-H and CDR-L are separately shown. CDR-H loops, CDR-L loops, and domain III are colored cyan, green, and gray, respectively. Residues that make key interactions are shown in the stick models. Hydrogen bonds are indicated by gray dotted lines.</p