Tm treatment induces phosphorylation of eIF2, reduces protein synthesis and selectively alters ribosome density of some transcripts.

<p>(A) Murine erythroblasts (line 15.4) were left untreated (-) or were treated for 90 min with Tm (2.5 μg/ml). Western blots with total cell lysates were probed for phosphorylated (anti P-S51 eIF2) and total eIF2. (B/C) Erythroblast samples as in (A) were processed for polyA+ RNA sequencing (RNAseq; B) or ribosome footprinting (RPF; C). We normalized and averaged sequence reads from 3 biologically independent experiments. For RNAseq We applied a threshold of, on average, 10 reads per condition (B). For RFP reads we applied a threshold of, on average, 1 read per condition (C). A statistical interaction model indicated differential ribosome density on 484 transcripts at a FDR <01%, indicated as red dots. (D) Depiction of 2Log FC ribo reads and RNA reads. Dashed gray line indicates the area where translation follows transcription. The fold-change (FC) in ribo reads (Tm-treated average reads/untreated average reads; Tm/Untr) was plotted against the FC Tm/Untr in RNA expression. Figures are based on data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193790#pone.0193790.s010" target="_blank">S2 Table</a>.