The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5′TOP sequence

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5′TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5′ UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5′TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signaling to ribosome biogenesis

Serratamolide is a hemolytic factor produced by Serratia marcescens

Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use. © 2012 Shanks et al

Serratamolide purification and verification of biological activity. A.

<p>Structure of serratamolide. <b>B.</b> HPLC trace of spent supernatants from a <i>swrW</i> mutant with either an empty vector (<i>swrW</i>+vector) or a <i>swrW</i> expression plasmid (<i>swrW</i>+p<i>swrW</i>). The expected peak for serratamolide is indicated by an arrow. <b>C.</b> Swarming motility of an <i>swrW</i> mutant treated with DMSO or purified serratamolide. This shows that the purified compound restores swarming motility as expected.</p

Isolation of <i>swrW</i> and its role in surfactant production and hemolysis. A.

<p>Sample genetic screen plate shows <i>crp</i> mutants with random transposon insertions. The white arrow indicates a colony deficient in secreted hemolysis production with a transposon insertion that mapped to the <i>swrW</i> gene. This image is illuminated from the back, so that the gold surface coloration is not apparent. <b>B.</b> Surface coloration of <i>crp swrW</i> double mutants is metallic gold compared to the red-orange color of the <i>crp</i> mutant. <b>C.</b> Surfactant zones (mm) measured from the colony to the maximum extent of the surfactant zone (n≥4 per genotype). Asterisk represents a statistically significant increase in surfactant zone compared to the WT (p<0.05) by ANOVA with Tukey’s post-test. <b>D.</b> Mutation of <i>swrW</i> reduced or eliminated the ability of laboratory strain Nima and three of five clinical keratitis isolates to make zones of hemolysis on blood agar plates. Representative images from reproducible experiments are shown.</p

A <em>Serratia marcescens</em> PigP Homolog Controls Prodigiosin Biosynthesis, Swarming Motility and Hemolysis and Is Regulated by cAMP-CRP and HexS

<div><p>Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen <i>Serratia marcescens</i> produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical <i>Serratia</i> species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in <i>S. marcescens</i>. Mutation of <i>pigP</i> in clinical, environmental, and laboratory strains of <i>S. marcescens</i> conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of <i>pigP</i>. This study suggests that PigP is important for the ability of <i>S. marcescens</i> to compete in the environment.</p> </div

Genetic evidence that serratamolide mediates hemolysis. A.

<p>Hemolysis and swarming by a mutant known to have elevated serratamolide production (<i>hexS</i>) is increased, and these phenotypes require SwrW. <b>B.</b> Elevated expression of a <i>swrW</i> promoter reporter in the <i>crp</i> mutant. Top, expression measured using a plasmid based-<i>tdtomato</i> reporter construct at t = 20 hrs. Asterisk indicates statistical significance (p<0.05) by the Student’s T-test. A representative experiment is shown (n = 4). Error bars indicate one standard deviation. Bottom, semi-quantitative RT-PCR analysis of RNA from WT and Δ<i>crp</i> mutant strains measured relative expression of <i>swrW</i> and internal standard 16S RNA from stationary phase cultures (OD₆₀₀ = ∼3.5). <b>C.</b> Arabinose-inducible expression of the <i>swrW</i> gene in an <i>swrW</i> transposon mutant strain restores hemolysis. <b>D.</b> Swarming motility defect of the <i>swrW</i> mutant is restored by induced expression of the <i>swrW</i> gene.</p

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis

Ribosomal defects perturb stem cell differentiation, causing diseases called ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discovered three RNA helicases are required for ribosome biogenesis and for Drosophila oogenesis. Loss of these helicases, which we named Aramis, Athos and Porthos, lead to aberrant stabilization of p53, cell cycle arrest and stalled GSC differentiation. Unexpectedly, Aramis is required for efficient translation of a cohort of mRNAs containing a 5’-Terminal-Oligo-Pyrimidine (TOP)-motif, including mRNAs that encode ribosomal proteins and a conserved p53 inhibitor, Novel Nucleolar protein 1 (Non1). The TOP-motif co-regulates the translation of growth-related mRNAs in mammals. As in mammals, the La-related protein co-regulates the translation of TOP-motif containing RNAs during Drosophila oogenesis. Thus, a previously unappreciated TOP-motif in Drosophila responds to reduced ribosome biogenesis to co-regulate the translation of ribosomal proteins and a p53 repressor, thus coupling ribosome biogenesis to GSC differentiation

PigP is necessary for hemolysis in laboratory and clinical isolates. A

<p>. Hemolytic strains grown on TSA plates with sheep blood show a zone of clearing around colonies indicative of hemolysis. Isogenic <i>pigP</i> mutant strains show highly reduced zones of hemolysis. <b>B.</b> The hemolysis defect of <i>pigP</i> mutants can be complemented by wild-type <i>pigP</i> on a plasmid (pMQ221); vector refers to pMQ132. <b>C.</b> Arabinose inducible expression of <i>swrW</i> is sufficient to restore hemolysis to the <i>pigP</i> mutant. The <i>swrW</i> gene was expressed from plasmid pMQ367 (p<i>swrW</i>), and vector refers to pMQ125. <b>D.</b> Mutation of <i>pigP</i> reduces the hyper-hemolytic phenotypes of <i>crp</i> and <i>hexS</i> mutants.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p