GoKawiilPlasmids
Cloning was performed either by Gibson assembly with the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs (NEB), E2621) or by site-directed mutagenesis by direct PCR with the KAPA HiFi HotStart Ready Mix (Roche, 07958935001) or Q5 Master Mix (NEB, M0492S). Plasmids were purified using the Plasmid Plus Midi Kit (QIAGEN, 12945) and verified by Sanger or nanopore sequencing. Constructs for insect cell expression were cloned into pFLN to facilitate bacmid generation in DH10EmBacY61.
Cell culture
All mammalian cells were cultured at 37 °C with 5% CO 2 . Expi293F cells (Gibco, A14527) were cultured in Expi293 expression media (Gibco, A1435102) and passaged 1:10 every 3 d to maintain a maximal cell density of 3 million cells per ml. K562 cells harbouring a miR-7-sensitive GFP reporter were a gift from J. Mendell10. K562 cells were cultured in RPMI 1640 (Gibco, 11875119) supplemented with 10% (v/v) fetal bovine serum (FBS) (Takara Bio, 631367) and passaged 1:4 every 3 d to maintain a maximal cell density of 800,000 cells per ml. Mouse embryonic fibroblast cells (MEFs) and HEK293FT cells were cultured in DMEM (Corning, 17-207-CV) with 10% FBS. Except in the case of contact inhibition for AGO2 intracellular co-IP and small-RNA-sequencing (sRNA-seq) experiments, described below, MEFs were passaged 1:4 or 1:8 as needed to maintain a maximal confluency of approximately 70%. HEK293FT cells were passaged 1:4 or 1:8 as needed to maintain a maximal confluency of approximately 70%. For passaging, adherent cells were dissociated using 0.25% trypsin–EDTA (Gibco, 25200114). For Expi293F cells, transfections were performed as described in the section ‘AGO–miRNA complexes for in vitro co-IP assays’. For K562 and HEK293FT cells, transfections were performed with Lipofectamine 2000 (Invitrogen, 11668019) and Opti-MEM (Gibco, 31985062) according to the manufacturer’s instructions. Drosophila S2 cells were cultured at 26 °C in Schneider’s Drosophila Medium (Gibco, 21720001) supplemented with 10% heat-inactivated FBS (Gibco, 16140071). S2 cells were passaged every 3–6 d to maintain an approximate concentration between 1 and 10 million cells per ml. For insect cell expressions, baculoviruses were generated from bacmids in Spodoptera frugiperda Sf9 insect cells (Thermo Fisher Scientific, 11496015) and used to infect Trichoplusia ni High-Five insect cells (Thermo Fisher Scientific, B85502) for expression. Cell lines were not authenticated. All cell lines tested negative for mycoplasma contamination upon arrival to the lab.
Proteins and RNAs
Proteins for in vitro ubiquitylation assays and for cryo-EM
Unless otherwise stated, all proteins are of human origin. Sequences of proteins and associated tags are listed in Supplementary Table 3. Genes were synthesized by TwistBiosciences or obtained as stated. Ubiquitin was expressed untagged and purified as described62. Cys-UB, in which ubiquitin carries an N-terminal cysteine for protein labelling, the lysine-less UB version (Cys-UB K0), NEDD8, UBE2L3, UBE2R2, UBE2M, ARIH1 and ARIH2 were expressed as GST-TEV or GST-3C fusions in BL21(DE3) RIL bacterial cells, as described35,39,63,64. Following glutathione-affinity pull-down (Cytiva, 17075605) and TEV protease cleavage, the protein was further purified by ion-exchange and size-exclusion chromatography. SrtA-5M, CUL3(NTD), CUL3(NTD, 1–384, I342R, L346D)-Avi and CUL3(∆N, NTD, 25–384, I342R, L346D)-Avi were expressed in BL21(DE3) RIL cells and purified by immobilized metal-affinity chromatography (Ni-INDIGO, Cube Biotech, 75105), followed by ion-exchange chromatography and size-exclusion chromatography, as described65,66. CUL2, CUL3, CUL5, GST-TEV-RBX1(5–108), GST-TEV(5–113) and CUL2(NTD, 1–380, L344K, V3776D)-Avi were expressed in insect cells using two baculoviruses for assembly of CUL–RBX complexes and purified following reported procedures35,63. CUL2–RBX1, CUL3–RBX1 and CUL5–RBX2 were neddylated and purified as described35,63.
ZSWIM8-Twin-Strep was cloned into pFLN. ELOB and ELOC were cloned into pFLN and assembled into pBig1a using biGBac Assembly61. ZSWIM8–ELOB–ELOC complex (hereafter referred to as ZSWIM8–ELOB/C) was expressed in insect cells using two baculoviruses for 3 d at 27 °C. Cells were resuspended in lysis buffer (50 mM HEPES, 300 mM NaCl and 10% (v/v) glycerol, pH 7.5) supplemented with protease inhibitors and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Cells were disrupted by sonication and cell debris was removed by centrifugation. ZSWIM8–ELOB/C was enriched using Twin-Strep affinity purification (Strep-Tactin, IBA Lifesciences, 2-1201-025) and eluted with lysis buffer supplemented with 3 mM desthiobiotin, followed by size-exclusion chromatography (HiLoad Superdex 200, Cytiva).
ZSWIM8mono was designed on the basis of our cryo-EM structure. We hypothesized that CUL3 binding to ZSWIM8 requires the presence of a folded D-domain. To functionally separate the requirement of ZSWIM8 dimerization for AGO2 binding and ubiquitylation from its impact on CUL3 binding, we designed a ZSWIM8 variant that retained a folded D-domain and retained binding to CUL3. We expected that transplanting a second copy of the D-domain into the native sequence would allow the D-domain to fold in cis and resemble the D-domain that is natively folded in trans. We inserted D-domain residues E222–P273 with two flanking GGSGGS linkers in between D-domain residues Q228 and R229 (Supplementary Table 2). AlphaFold3 structure predictions supported the expected assembly of the D-domain.
For structural studies and stepwise ubiquitin transfer assays, His 6 -TEV-AGO2 was cloned into pFLN and expressed in insect cells. AGO was purified following an adapted purification protocol67. In brief, insect cells were resuspended in 50 mM Tris, 100 mM KCl and 5 mM dithiothreitol (DTT), pH 8.0. Following sonication, polyethylenimine was added to a concentration of 0.2% (v/v), mixed by inversion and immediately centrifuged to remove cell debris. AGO was enriched by immobilized metal-affinity chromatography (Ni-INDIGO, Cube Biotech, 75105). Following affinity chromatography, a nucleic-acid-free AGO fraction (as judged by 260/280 ratio) was isolated by cation-exchange chromatography (Source 15S or MonoS, Cytiva) with a gradient of 0–500 mM KCl in 20 mM Tris, pH 8.0. Nucleic-acid-free AGO was incubated with synthetic single-stranded miRNA (Metabion, IDT; Supplementary Table 4) at room temperature for 30 min. Excess miRNA was removed by size-exclusion chromatography in 25 mM HEPES, 150 mM NaCl and 1 mM TCEP, pH 7.8 (Superdex 200, Cytiva).
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