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Additional file 1: Additional figures and figure legends in a single pdf file with: Figure S1. Comparison of mRNAs that are more or less soluble. Figure S2. mRNAs that are more or less soluble upon Not1 and Not4 depletion are enriched for different GO-terms. Figure S3. Comparison of RDO changes between mutants and RNA pools. Figure S4. Quality control of the Not4 PAR-CLIP. Table S5. List of strains and plasmids.

Additional file 7. Review history.

Additional file 6. A separate pdf file entitled: Uncropped blots. Related to Figure S2A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

Figure S1. Biological replicates of the comparative tRNA microarrays are highly reproducible. a-e Data for HBE (a), organoids (b), CFBE41o- (c), FRT (d) and HNE from a male (NL124) or female (NL117) individual (e) are shown as fold-enrichment (gradient ruler at the bottom) of tRNAs compared with HEK293 cells. Global reproducibility between each two replicates was assessed by Kolmogorov-Smirnov test (for all arrays pâ â Ľâ 0.9, i.e. very similar). The reproducibility for each tRNA probe was assessed by variability analysis of each two replicates and is presented as coefficient of variance. tRNA isoacceptors are depicted with their anticodon and corresponding amino acid; Meti-CAU, initiator tRNAMet pairing to the AUG codon. Figure S2. Correlation of tRNA abundance between tested models. a Pairwise correlation of tRNA isoacceptor abundancies. For iPSCs, only the zero time point was considered. R, Pearson correlation coefficient. b Correlation of tRNA isoacceptor abundancies for iPCSs over the course of differentiation. Stem cells at days 5, 10, 15 and 21 (gradient ruler) were compared to non-differentiated cells at time zero. Figure S3. Standard deviations of computed ribosome occupancies per codon among models. In these calculations, only the zero time point was considered for iPSCs. (DOCX 1932 kb)

Overview of bioinformatics predictions for Nog1. (PDF 110 kb)

E. coli strains and plasmids. (PDF 116 kb)

Overview of bioinformatics predictions for Nog1. (PDF 110 kb)

E. coli strains and plasmids. (PDF 116 kb)

Full description of the metabolomics measurements. (PDF 125 kb)