Fig. 2

Ribosome profiling identifies mRNAs regulated by the Ccr4-Not complex at the level of translation. A Western blot confirms siRNA knockdown of CNOT1. Vinculin is used as a loading control. B Polysome gradient profiles for samples treated with control or CNOT1-targeting siRNA. C There are groups of mRNAs with distinct changes in ribosome occupancy and mRNA abundance when CNOT1 is depleted. Log2 fold change of RPFs and RNA following CNOT1 depletion were determined using DESeq2 independently for each library type. Log2 translational efficiency (TE) was determined by log2FC RPF − log2FC RNA; a threshold of log2FC TE > 0.2 was used to categorize mRNAs as having increased TE and a log2FC TE < − 0.2 for mRNAs with decreased TE. No TE change was classified by a log2TE < 0.1 & > − 0.1. The table shows the number of mRNAs present in each group. D–F qPCR along gradient fractions from an independent experiment with and without CNOT1 depletion (n = 1 shown, n = 2 is shown in Additional File 1: Fig. S6). D Validation of the increased TE after CNOT1 depletion of POLB and HDHD3 from the group of mRNAs identified in (C) (red). In gray is the control and in red is the CNOT1 siRNA treated. E Validation of the unchanged TE after CNOT1 depletion for DENR and INTS7 from the group of mRNAs identified in (C) (yellow). In gray is the control and in yellow is the CNOT1 siRNA treated. F Validation of the decreased TE after CNOT1 depletion of GIT1 and POLR3E from the group of mRNAs identified in (C, blue). In gray is the control and in blue is the CNOT1 siRNA treated