Inactivation of Ded1 evokes accumulation in 48S PICs of LUC reporter mRNA with a stable cap-distal SL insertion. (A,B) WT and ded1-cs strains harboring LUC reporters described in Figure 5A,B, with a 91-nt 5′ UTR or containing the −3.7 kcal/mol SL, were cultured as in Figure 4B and cells were cross-linked with 2% HCHO for 1 h prior to harvesting. WCEs were resolved by sedimentation through 10%–50% sucrose gradients, and fractions were collected while scanning at 254 nm. (A) Representative polysome profiles. (B) Gradient fractions were spiked with equal amounts of control RNA, total RNA extracted, and abundance of LUC reporter (relative to control) mRNA quantified by qRT-PCR, conducting three technical replicates for each fraction. Results from biological replicates ±SEM are shown.
