Studies on the Coordination of Ribosomal Protein Assembly Events Involved in Prosessing and Stabiliz

Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

International audienceInnate immune recognition of the major human-specific Gram-positive pathogen Strepto-coccus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2-and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon in-ternalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 −/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms

Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli

Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin–antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine–cytosine–adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5′-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes

Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli

The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12

A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed

Changes in r-protein assembly states of early LSU precursors after <i>in vivo</i> depletion of LSU r-proteins required for early LSU pre-rRNA processing.

<p>The dataset generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g003" target="_blank">Fig 3</a> was analyzed in respect to changes in r-protein levels in Noc2-TAP fractions isolated from wild type cells or from r-protein expression mutants. Observed changes in levels of r-proteins and the results of clustering analyses are visualized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g003" target="_blank">Fig 3B</a>. Groups of r-proteins mentioned in the text are highlighted by bars on the right. Only r-proteins which were identified in at least 70% of the 17 pairwise comparisons were included in these analyses. The r-proteins whose expression was shut down in the respective experiment (“mutant <i>versus</i> wild type”) are highlighted by a red box.</p

Interactions of dII/dVI cluster r-proteins with rRNA in mature ribosomes.

<p>The yeast LSU is shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g001" target="_blank">Fig 1</a> viewed from the solvent exposed side in <b>A)</b>, <b>C)</b> and <b>E)</b>. It is rotated by approximately 90 degree around a horizontal axis in <b>B)</b>, <b>D)</b> and <b>F)</b>. In <b>A)</b>–<b>F)</b> LSU rRNA domain II is colored in yellow, LSU rRNA expansion segment 7 in orange, LSU rRNA domain VI in blue, 5.8S rRNA in red and other parts of the LSU rRNA in grey. In <b>A)</b> and <b>B)</b> dII/dVI cluster r-proteins are the only r-proteins shown and colored in shades of green. In <b>C)</b> and <b>D)</b> only rRNA is visualized and in <b>E)</b> and <b>F)</b> rpL16/uL13 is the only r-protein shown, with its globular domain in light green and its C-terminal clamp-like domain in dark green.</p