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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 the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine ‘tail’ is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.

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)

International audience; Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1+ late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3+/LAMP2+/LAMP1− endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.

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)

Background Gene duplication is believed to be the classical way to form novel genes, but overprinting may be an important alternative. Overprinting allows entirely novel proteins to evolve de novo, i.e., formerly non-coding open reading frames within functional genes become expressed. Only three cases have been described for Escherichia coli. Here, a fourth example is presented. Results RNA sequencing revealed an open reading frame weakly transcribed in cow dung, coding for 101 residues and embedded completely in the −2 reading frame of citC in enterohemorrhagic E. coli. This gene is designated novel overlapping gene, nog1. The promoter region fused to gfp exhibits specific activities and 5’ rapid amplification of cDNA ends indicated the transcriptional start 40-bp upstream of the start codon. nog1 was strand-specifically arrested in translation by a nonsense mutation silent in citC. This Nog1-mutant showed a phenotype in competitive growth against wild type in the presence of MgCl2. Small differences in metabolite concentrations were also found. Bioinformatic analyses propose Nog1 to be inner membrane-bound and to possess at least one membrane-spanning domain. A phylogenetic analysis suggests that the orphan gene nog1 arose by overprinting after Escherichia/Shigella separated from the other γ-proteobacteria. Conclusions Since nog1 is of recent origin, non-essential, short, weakly expressed and only marginally involved in E. coli’s central metabolism, we propose that this gene is in an initial stage of evolution. While we present specific experimental evidence for the existence of a fourth overlapping gene in enterohemorrhagic E. coli, we believe that this may be an initial finding only and overlapping genes in bacteria may be more common than is currently assumed by microbiologists. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0558-z) contains supplementary material, which is available to authorized users.

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