Transfection

For siRNA transfection, cells were plated at 2 × 10⁶ cells per 10 cm plate, cultured for 12 hr, and transfected with 15 pmol total siRNAs and 45 µl Lipofectamine RNAiMAX (Thermo Fisher, 13778500) in 1 ml Opti-MEM media (Thermo Fisher, 31985062) according to manufacturer’s protocol. Cells were split 1:4 onto new plates 24 hr after transfection and then collected for analysis 48 hr (Figure 4—figure supplement 2A-B, Figure 4—figure supplement 3, Figures 5–6, Figure 6—figure supplement 1) or 72 hr (Figure 4, Figure 4—figure supplement 1 and Figure 4—figure supplement 2C–G) after transfection. For rescue of PABPC-knockdown, cells were plated at 0.6 × 10⁶ cells per well in 6-well plates, and transfected with 2.5 pmol total siRNAs and 7.5 µl Lipofectamine RNAiMAX in 250 µl Opti-MEM media. After 24 hr, cells were transfected with 1 µg DNA and 3 µl FuGENE HD (Promega, E2311) in 150 µl Opti-MEM media according to manufacturer’s protocol, and collected for analysis 40 hr after DNA transfection. For other DNA plasmid transfections, cells were plated at 0.6 × 10⁶ cells per well in 6-well plates, cultured for 24 hr, transfected with 1 µg DNA and 3 µl FuGENE HD (Promega, E2311) in 150 µl Opti-MEM media according to manufacturer’s protocol. siRNAs were all purchased from Dharmacon, including siControl (D-001810-10-05), human siPABPC1 (L-019598-00-0005), human siPABPC4 (L-011528-01-0005), mouse siPabpc1 (L-060385-00-0005), human siTUT4 (L-021797-01-0005), and human siTUT7 (L-026009-00-0005). Plasmids and their sequence information will be available at Addgene.