Fig. 3

ER-targeted mRNAs are translationally downregulated following CNOT1 depletion. A All mRNAs were ordered by the log2FC TE (siCNOT1/siControl) and gene set enrichment analysis conducted on the ranked list using the fgsea R package for cellular components. B Western blot confirming lysate fractionation into ER and cytosol with and without the depletion of CNOT1. C k-means clustering of mRNAs based on their abundance (CPM) in the ER and cytosolic fractions defines two groups of mRNAs based on their predominant localization. D mRNAs with decreased TE when CNOT1 is depleted (identified in Fig. 2C) are enriched for targeting to the ER in control conditions. E Median change in ribosome occupancy (normalized for mRNA abundance) assessed across the CDS for the groups of mRNAs identified in Fig. 2C. F ER-targeted mRNAs globally decrease translationally efficiency following CNOT1 depletion. G log2FC RNA in the cytosolic fraction between CNOT1 depleted and control conditions. H log2FC RNA in the ER fraction between CNOT1 depleted and control conditions shows a reduction in mRNAs that are targeted to the ER in control conditions localizing to the ER after CNOT1 depletion. I Displayed is the protein production change (siCNOT1/siControl) for mRNAs predominantly localized to the cytosol or the ER. For (F–I), significance was determined using the Kruskal-Wallis test. J Median change in ribosome occupancy (normalized for mRNA abundance) assessed across the CDS for the mRNAs with decreased TE after CNOT1 depletion separated into two groups based on the predicted presence of a signal sequence (determined using SignalP [91]). 157/899 mRNAs were predicted to contain a signal sequence. K Predicted location of the signal peptidase cleavage site in the group of mRNAs with a predicted signal sequence (determined using SignalP [91])