Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized

Reciprocal regulation of protein synthesis and carbon metabolism for thylakoid membrane biogenesis

Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes

Literaturvermittlung in der sozialdemokratischen Presse: 1876-1933

Mittels einer Konfigurationsfrequenzanalyse untersucht die Autorin für den Zeitraum von 1876 bis 1933 die theoretischen Konzepte der Sozialdemokratischen Partei Deutschlands über die Vermittlung von Literatur in der Arbeiterpresse, die Praxis der Literaturvermittlung sowie die Literaturrezeption in Bezug auf Autoren, deren Werke und der sozialdemokratischen Verwertungsstrategien anhand der Tageszeitungen 'Berliner Volksblatt' und 'Vorwärts', der illustrierten Wochenbeilage 'Neue Welt' und der Wochenzeitschrift 'In freien Stunden'. Allgemein konstatiert die Verfasserin, daß es der sozialdemokratischen Presse nicht gelungen ist, eine der proletarischen Lebenswelt entsprechende Literaturpolitik zu entwickeln und festzuschreiben. Da die vorliegende Studie einen Ausschnitt aus der Dissertation der Autorin darstellt, werden Einzelergebnisse lediglich zum Wochenblatt 'In Freien Stunden' vorgestellt. Dabei ermittelt die Verfasserin Präsentation und Rezeption bürgerlicher, meist verstorbener Erzähler der Weltliteratur insbesondere aus dem deutschsprachigen und westeuropäischen Raum im Nachdruck. Diese Verwertung steht nach Ansicht der Autorin in krassem Gegensatz zur Programmatik des Presseorgans, eine der Arbeiterkultur gemäße und sie fördernde Literatur zu vermitteln. Begründet wird dies von der Verfasserin mit der strikten Trennung von Kunst und Politik innerhalb der Sozialdemokratie und der Instrumentalisierung der Literatur für die Köderung neuer Abonnenten für die Parteipresse. (RS)'We investigate the theoretical concepts of the Social Democratic Party concerning the communication of literature in the worker's press, the practice of literature communication and the reception of literature. Different types of press were used for content analysis: the 'Berliner Volksblatt' (1884-1890) and the 'Vorwärts' (1891-1933) represent the daily press; the 'Neue Welt' (1876-1887 and 1892-1919) represents the illustrated supplement and 'In Freien Stunden' (1897-1918/19) the fiction weekly. This report deals mainly with the results optained for the fiction weekly. Configuration frequency analyses show that different concepts of literature communication were applied to the press organs. These differences concern the selection strategies of the published authors, of the literary products and of exploitation strategies. Each organ can thus defined by particular characteristics and by typologically relevant configurations of characteristics. These are for the fiction weekly 'In Freien Stunden': non-coincidentally frequent publication of bourgeois narrative writers of world literature, hardly any socialdemocratic author; non-coincidentally frequent publication of works no longer protected by copyright (authors dead for more than 30 years!); no connection to contemporary bourgeois literary trends. Non-coincidentally often publication of German-language and West-European authors (French and British), hardly ever Scandinavian and Eastern-European authors. The typologically most relevant and efficient characteristics of authors was shown to be their affiliation to the canon of world literature and the fact that at the time of publication they had been dead for a long time.' (author's abstract

Mixtures of rare earth elements show antagonistic interactions in Chlamydomonas reinhardtii

In order to better understand the environmental risks of the rare earth elements (REEs), it is necessary to determine their fate and biological effects under environmentally relevant conditions (e.g. at low concentrations, REE mixtures). Here, the unicellular freshwater microalga, Chlamydomonas reinhardtii, was exposed for 2 h to one of three soluble REEs (Ce, Tm, Y) salts at 0.5 μM or to an equimolar mixture of these REEs. RNA sequencing revealed common biological effects among the REEs. Known functions of the differentially expressed genes support effects of REEs on protein processing in the endoplasmic reticulum, phosphate transport and the ho- meostasis of Fe and Ca. The only stress response detected was related to protein misfolding in the endoplasmic reticulum. When the REEs were applied as a mixture, antagonistic effects were overwhelmingly observed with transcriptomic results suggesting that the REEs were initially competing with each other for bio-uptake. Metal biouptake results were consistent with this interpretation. These results suggest that the approach of government agencies to regulate the REEs using biological effects data from single metal exposures may be a largely con- servative approach

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38–40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA

The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

Photosystem II is the first of two light-driven oxidoreductase complexes in oxygenic photosynthesis. The biogenesis of photosystem II requires the synthesis of polypeptide subunits encoded by the genomes in the chloroplast and the nucleus. In the chloroplast of the green alga Chlamydomonas reinhardtii, the synthesis of each subunit requires interactions between the 5′ UTR of the mRNA encoding it and gene-specific translation factors. Here, we analyze the sequences and structures in the 5′ UTR of the psbC mRNA, which are known to be required to promote translation and genetic interaction with TBC1, a nuclear gene required specifically for psbC translation. Results of enzymatic probing in vitro and chemical probing in vivo and in vitro support three secondary structures and reveal that one participates in a pseudoknot structure. Analyses of the effects of mutations affecting pseudoknot sequences, by structural mapping and thermal gradient gel electrophoresis, reveal that flexibility at the base of the major stem-loop is required for translation and higher order RNA conformation, and suggest that this conformation is stabilized by TBC1. This RNA pseudoknot tertiary structure is analogous to the internal ribosome entry sites that promote translation of certain viruses and cellular mRNAs in the nuclear-cytoplasmic systems of eukaryotes

Biological impacts of Ce nanoparticles with different surface coatings as revealed by RNA-Seq in Chlamydomonas reinhardtii

In order to better understand the risks of engineered nanoparticles (ENPs), it is necessary to determine their fate and biological effects under realistic exposure scenarios (e.g. low ENP concentrations). RNA-Seq was deployed to characterize the relative biological impacts of three small Ce ENPs (i.e. nominal size < 20 nm, 70 μg L−1 Ce), with different coating properties (i.e. uncoated, citrate or poly-acrylic acid coated), towards a unicellular freshwater microalga, Chlamydomonas reinhardtii. After 2 h exposition at pH 7.0, distinct differences in tran- scriptomic effects were observed when comparing ionic Ce and Ce ENPs. Notably, Ce ENPs specifically modu- lated mRNA levels of genes related to the ubiquitin-proteasome system and to flagella structure. Compared to control conditions, transcriptomic effects induced by the citrate coated Ce ENPs were rather limited, as only 23 genes were differentially expressed by this treatment (Log2FC > |1.0|, padj < 0.001); compared to uncoated Ce ENPs (688); polyacrylic coated Ce ENPs (315) or a similar concentration of ionic Ce (138). Somewhat surpris- ingly, similar changes in the algal transcriptomes were observed for treatments with poly-acrylic acid coated Ce ENPs (mainly Ce(III), little dissolution) and uncoated Ce ENPs (mainly Ce(IV) atoms, largely agglomerated) (Log2FC > |1.0|, padj < 0.001). For the moderate exposure concentrations examined here, toxicity appeared to be minimal for both ionic Ce and Ce ENPs. Nonetheless, an important number of genes could not be assigned to a biological pathway. The study gives important insights with respect to the role of particle surface coatings on biological effects, the mechanisms of interaction of Ce ENP with a green alga, in addition to identifying several useful transcriptomic biomarkers of Ce ENP exposure

Protein targeting to subcellular organelles via mRNA localization

Cells have complex membranous organelles for the compartmentalization and regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or translocation to an organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes to the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane-embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile, research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is encoded by cis-acting sequences in the mRNA (“Zipcodes”) that interact with localization factors and, in many cases, are transported by molecular motors on cytoskeletal filaments. Recently, evidence has been found for this “mRNA-based” mechanism in organelleproteintargeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNAlocalization in proteintargeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids