A comprehensive analysis of the importance of translation initiation factors for Haloferax volcanii applying deletion and conditional depletion mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity Evidence From Mouse and Human Studies

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2-or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/beta-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=1215) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/\CXCR4, which triggered Akt/WNT/beta-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

Haloferax volcanii, a prokaryotic species that does not use the Shine Dalgarno mechanism for translation initiation at 5′-UTRs

It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5′-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20–30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5′-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5′UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5′-UTRs. A genome analysis revealed that while the number of SD motifs in 5′-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons

Occurrence of SD motifs upstream of distal genes in operons.

<p>108 nt upstream of all genes with a distance of less than 10 nt to the preceding were retrieved from the genome of <i>H. volcanii</i>. The sequences were searched for the occurrence of motifs with a 5–8 nt match to the SD motif GGAGGUGA. A. the occurrence of motifs with matches of at least 6 nt (purple curve), at least 7 nt (blue curve), and 8 nt (red curve) are shown. B. smoothed curves (3 nt average) are shown for the occurrence of exactly 5 nt (green curve), exactly 6 nt (purple curve), exactly 7 nt (blue curve), and exactly 8 nt (red curve).</p

Spacing between the 8 nt SD motif to the start codon of the downstream gene.

<p>In 31 cases the extended 8 nt SD motif was found to be localized at the 3′-end near the stop codon. The sequences around the SD motif were retrieved from the genome sequence of <i>H. volcanii</i> and the gene identifier (HVO numbers) and sequences are shown. It turned out that all 31 genes were proximal genes in operons and were closely followed by a downstream gene. The SD motif and the start codon of the downstream gene are shown in red, and the stop codon of the upstream gene is shown in bold.</p

Translational efficiencies of the consecutive SD mutant series at reduced growth rates.

<p>For the comparative analysis of translational efficiencies <i>H. volcanii</i> cultures of pPK10-pPK18 and pNP10 negative control (NC) were grown to mid-exponential growth phase at the reduced temperature of 30°C (A, B) or with acetate as carbon and energy source (C, D). A. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s003" target="_blank">Table S3</a>. B. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3). C. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s004" target="_blank">Table S4</a>. D. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3).</p

Translational efficiencies of different transcripts with and without an SD sequence.

<p>For the comparative analysis of translational efficiencies <i>H. volcanii</i> cultures of pPK19-pPK22 were grown to mid-exponential growth phase under standard conditions. A. Schematic overview of the reporter gene constructs pPK19-pPK22. The native SD motif of the <i>sod</i> gene (green and underlined) was replaced by three randomly generated unrelated sequences in the mutants pPK20-pPK22. A second leaderless translation start site AUG was introduced at the 5′-end of the 51 nt elongated <i>sod</i> 5′-UTR (blue). B. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s005" target="_blank">Table S5</a>. C. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3). Results of proteins originating from the leaderless start codon are shown in blue, results of proteins originating from the <i>sod</i> start codon are shown in green.</p

Translational efficiencies of a consecutive SD mutant series under standard conditions.

<p>For the comparative analysis of translational efficiencies H. volcanii cultures of pPK10-pPK18 and pNP10 negative control (without hdrA) were grown to mid-exponential growth phase under standard conditions (2.1 M NaCl, 42°C). A. Schematic overview of the reporter gene constructs pPK10-pPK18. The SD sequence (green and underlined) was mutated as indicated. B. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s002" target="_blank">Table S2</a>. C. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3).</p

Bioinformatic analysis of the <i>H. volcanii</i> genome.

<p>A. Sequence logo of 5′ regions of 3025 putative monocistronic genes or proximal genes in operons with an intergenic distance at least 40 nt to the adjacent gene. B. Sequence logo of 5′ regions of 791 putative distal genes in operons with an intergenic distance of less than 10 nt to the adjacent gene.</p