Extended Data Figure 5: Time course of DENR knockdown reveals that changes in stuORF translation precede global changes in polysome profiles or ribosome levels.
From: DENR–MCT-1 promotes translation re-initiation downstream of uORFs to control tissue growth
a, Translation of a stuORF reporter (bearing 1× ATGTAA, as in Fig. 3) progressively drops upon DENR knockdown from 1–4 days after treatment with dsRNA. A significant drop in translation can be observed already 2 and 3 days after DENR knockdown. In contrast, a control reporter is unperturbed. b, b′, DENR protein levels 2 (b) and 3 (b′) days after knockdown in S2 cells, assessed by immunoblotting. c, c′, Polysome profile of S2 cells treated for 2 (c) or 3 (c′) days with DENR dsRNA (red traces) shows no significant drop in polysome fractions compared to cells treated with control (GFP) dsRNA (grey traces), suggesting that the drop in polysome levels observed at later time points (for example, Extended Data Fig. 1) is in vivo secondary consequences (for example, due to reduced translation of stuORF-containing transcripts such as Insulin Receptor, leading to a global reduction in translation rates). d, d′, Quantification of 18S RNA, 28S RNA and initiator tRNA levels in S2 cells treated with dsRNA for 2 (d) or 4 (d′) days shows little to no increase upon DENR knockdown compared to control.
