Molecular basis of polyadenylated RNA fate determination in the nucleus

DNA 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). …