GoKawiilDNA sequences
All oligonucleotide plasmid vectors are annotated in Supplementary Table 6.
Purification of UAP56 and UAP56(Δ1–43)
His-tagged UAP56 constructs (10×His–3C–UAP56 or 10×His–3C–UAP56(Δ1–43), residues 44–428) were expressed in Escherichia coli BL21 DE3 RIL using autoinduction medium at 37 °C for 16 h. Following collection, cells were resuspended in lysis buffer (25 mM HEPES pH 7.9, 5% glycerol, 300 mM NaCl, 20 mM imidazole, 0.05% Tween-20, and protease inhibitors), disrupted via sonication, and clarified by centrifugation. The supernatant was sequentially filtered through 1-µm and 0.45-µm filters before affinity purification on a HisTrap HP 5 ml column (Cytiva), equilibrated in buffer A (25 mM HEPES pH 7.9, 5% glycerol, 300 mM NaCl, 20 mM imidazole). After washing with buffer A supplemented with 70 mM imidazole, bound proteins were eluted using a linear gradient of imidazole (70–200 mM in buffer A). Peak fractions were diluted in buffer B (25 mM HEPES pH 7.9, 5% glycerol, 1 mM DTT) to reduce the NaCl concentration to 100 mM and subsequently subjected to anion-exchange chromatography on a HiTrapQ 5 ml column (Cytiva), pre-equilibrated with buffer B. Elution was performed with a linear NaCl gradient (100–500 mM). Fractions containing UAP56 were concentrated and further purified via size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (Cytiva), equilibrated in buffer C (25 mM HEPES pH 7.9, 5% glycerol, 100 mM NaCl, 1 mM DTT). Peak fractions containing the purified protein were pooled, concentrated, flash-frozen, and stored at −80 °C.
Purification of LENG8–PSM and SAC3D1–PSM
Expression constructs encoding LENG8–PSM (10×His–MBP–LENG8491–800, 3×V5–PCID2, SEM1), SAC3D1–PSM (10×His–MBP–SAC3D148–404, 3×V5–PCID2, SEM1), SAC3D1–PCID2-UAP56–UCM–N-UBM–SEM1 (10×His–MBP–SAC3D148–404, 3×V5–PCID2–UAP56–UCM–N-UBM, SEM1), LENG8–PCID2–UAP56–N-UBM, SEM1 (10×His–MBP–LENG8491–800, 3×V5–PCID2–UAP56–N-UBM, SEM1) and their respective mutants were introduced into E. coli BL21 DE3 RIL (UCM is a UAP56-clamping motif). Cultures were grown in LB medium at 37 °C to OD600 ~1.0, at which point expression was induced with 0.5 mM IPTG, followed by overnight incubation at 18 °C. Cells were collected, lysed by sonication, and clarified by centrifugation. The supernatant was filtered (1 µm and 0.45 µm) and loaded onto a HisTrap HP 5 ml column equilibrated with buffer A, followed by washing and elution using a linear imidazole gradient up to 300 mM. Peak fractions were diluted to 50 mM NaCl in buffer B and subjected to anion-exchange purification on a HiTrapQ HP 5 ml column. After washing, complexes were eluted with a NaCl gradient (100–500 mM). Size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (Cytiva) in buffer C containing 250 mM NaCl yielded the final purified complex, which was concentrated, flash-frozen, and stored at −80 °C.
Recombinant EIF4A3 was purified as described previously16.
His-tagged DDX19 was expressed in E. coli BL21 DE3 RIL using LB medium, induced with 0.5 mM IPTG and expressed at 37 °C for 3 h. Following collection, cells were resuspended in lysis buffer (25 mM HEPES pH 7.9, 5% glycerol, 300 mM NaCl, 20 mM imidazole, and protease inhibitors), disrupted via sonication, and the lysate was cleared by centrifugation. The supernatant was sequentially filtered through 1-µm and 0.45-µm filters before affinity purification on a HisTrap HP 5 ml column (Cytiva), equilibrated in buffer A. The column was washed with buffer A containing 30 mM imidazole and bound proteins were eluted using a linear gradient of imidazole (50–300 mM). The peak fractions were incubated with 3C protease to cleave off the tag, and after 3C cleavage the peak fractions were diluted in buffer B to reduce the NaCl concentration to 50 mM, filtered through a 0.22-µm filter and next subjected to anion-exchange chromatography on a HiTrapQ 5 ml column (Cytiva), pre-equilibrated with buffer B supplemented with 50 mM NaCl. The column was washed with buffer B supplemented with 50 mM NaCl following sample loading. Elution was performed with a linear NaCl gradient (50–500 mM). Peak fractions containing DDX19 were concentrated and further purified via size-exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (Cytiva), equilibrated in buffer C. Peak fractions containing the purified protein were pooled, concentrated, flash-frozen, and stored at −80 °C.
Analytical gel filtration
For each purified protein or complex an aliquot of 62.5 μg was loaded onto a Superdex 200 Increase 5/150 column (Cytiva), equilibrated in the respective gel filtration buffers. Peak fractions were analysed via SDS–PAGE (4–12% gradient) and visualized by Coomassie staining.
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