The DEAD-box protein MEL-46 is required in the germ line of the nematode Caenorhabditis elegans

Background: In the hermaphrodite of the nematode Caenorhabditis elegans, the first germ cells differentiate as sperm. Later the germ line switches to the production of oocytes. This process requires the activity of a genetic regulatory network that includes among others the fem, fog and mog genes. The function of some of these genes is germline specific while others also act in somatic tissues. DEAD box proteins have been shown to be involved in the control of gene expression at different steps such as transcription and pre-mRNA processing.Results: We show that the Caenorhabditis elegans gene mel-46 (maternal effect lethal) encodes a DEAD box protein that is related to the mammalian DDX20/Gemin3/DP103 genes. mel-46 is expressed throughout development and mutations in mel-46 display defects at multiple developmental stages. Here we focus on the role of mel-46 in the hermaphrodite germ line. mel-46(yt5) mutant hermaphrodites are partially penetrant sterile and fully penetrant maternal effect lethal. The germ line of mutants shows variable defects in oogenesis. Further, mel-46(yt5) suppresses the complete feminization caused by mutations in fog-2 and fem-3, two genes that are at the top and the center, respectively, of the genetic germline sex determining cascade, but not fog-1 that is at the bottom of this cascade.Conclusion: The C. elegans gene mel-46 encodes a DEAD box protein that is required maternally for early embryogenesis and zygotically for postembryonic development. In the germ line, it is required for proper oogenesis. Although it interacts genetically with genes of the germline sex determination machinery its primary function appears to be in oocyte differentiation rather than sex determination

Screening fungi isolated from historic Discovery Hut on Ross Island, Antarctica for cellulose degradation

To survive in Antarctica, early explorers of Antarctica's Heroic Age erected wooden buildings and brought in large quantities of supplies. The introduction of wood and other organic materials may have provided new nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials. From 30 samples taken from Discovery Hut, 156 filamentous fungi were isolated on selective media. Of these, 108 were screened for hydrolytic activity on carboxymethyl cellulose, of which 29 demonstrated activities. Endo-1, 4-β-glucanase activity was confirmed in the extracellular supernatant from seven isolates when grown at 4°C, and also when they were grown at 15°C. Cladosporium oxysporum and Geomyces sp. were shown to grow on a variety of synthetic cellulose substrates and to use cellulose as a nutrient source at temperate and cold temperatures. The research findings from the present study demonstrate that Antarctic filamentous fungi isolated from a variety of substrates (wood, straw, and food stuffs) are capable of cellulose degradation and can grow well at low temperatures

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult <i>C. elegans</i> Germ Line

<div><p>To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult <i>Caenorhabditis elegans</i> reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that <i>gld-4</i> activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with <i>glp-1</i> mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of <i>glp-1</i> mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.</p></div

<i>glp-1</i> mRNA associates with GLD-4 and is a likely target of poly(A) tail extension and translational activation.

<p>(A,B) RNA-coimmunoprecipitation experiments (RIPs) of GLD-4 and GLS-1 proteins specifically enrich <i>glp-1</i> mRNA and the positive control <i>gld-1</i> mRNA. <i>eft-3</i> and <i>rpl-11.1</i> mRNA served as negative controls. (A) A representative ethidium bromide-stained agarose gel of semiquantitative RT-PCR products from three independent biological replicates. (B) Quantitative RT-PCR measurements of three additional RIPs. Error bars are SEM. ***, p<0.001; **, p<0.01; n.s., not significant (Student's t-test). (C,D) Translational efficiency of <i>glp-1</i> mRNA depends on <i>gld-4</i> activity. The data are representative of three independent biological experiments. (C) Polysome gradient. Top is to the right; grey peaks represent optical density read of 258 nm; the peaks of the large ribosomal subunit (60S), monosomes (80S), and polysomes are indicated. Relative <i>glp-1</i> mRNA levels are lower in polysome fractions of <i>gld-4</i>(RNAi) as measured by RT-qPCR. (D) Quantification and comparison of <i>glp-1</i> mRNA in pooled polysomal (polys.) and non-polysomal (non-polys.) fractions. Each measurement was normalized to an internal spike-in control (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#s4" target="_blank">Materials and Methods</a>). Error bars are SEM. *, p<0.05; n.s., not significant (Student's t-test). (E,F) poly(A) tails of <i>glp-1</i> mRNA are reduced upon <i>gld-4</i>(RNAi). (E) Representative PAT assay (n = 2) of the <i>glp-1</i> mRNA material from (C) and the gradient input material. Nucleotide size marker to the left. Lane 7 reflects a 3′UTR with a strongly reduced poly(A) tail (pA) after RNAase H and oligo dT treatment (H/dT). (F) Line scans of PAT assay from (E).</p

Differential GLD-4 and GLD-2 expression in the proliferative zone is FBF dependent.

<p>(A) GLD-4 expression is equal across the distal germ line. GLD-2 intensities increase from low-to-high in a distal-to-proximal manner. Extruded gonads of indicated genotype stained with DAPI, α-GLD-2, α-GLD-4, and α-GLH-2 as a positive tissue penetration control (not shown). Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. (B,C) Distal GLD-2 expression is repressed by <i>fbf</i> activity. (B) Example of an <i>fbf</i>(RNAi) immunostained extruded gonad. For the complete RNAi experiment see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647.s001" target="_blank">Figure S1</a>. (C) Quantification of the complete <i>fbf</i>(RNAi) experiment. Four different regions of nine germ lines per genotype were analyzed in their median, primarily cytoplasmic area. Error bars are SEM. ***, p<0.001; **, p<0.01; *, p<0.05; bars without indicated p value are statistically not significant (Student's t-test). (D, E) FBF binds specifically to at least one of the five predicted sequence elements in the <i>gld-2</i> 3′UTR. (D) Schematic drawing of the 1094 nt long <i>gld-2</i> 3′UTR. Sequence alignment of FBF-binding element consensus (FBE cons.) sequence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Lamont1" target="_blank">[14]</a> and the conserved FBE4 element in three <i>Caenorhabditis</i> species: <i>ce</i>, <i>C. elegans</i>; <i>cb</i>, <i>C. briggsae</i>; <i>cr</i>, <i>C. remanei</i>. pA indicates beginning of the poly(A) tail. (E) Yeast three-hybrid assay. RNA hybrid and Gal4-protein fusions are indicated. FBF-1, FBF-2 and PUF-5 belong to same RNA-binding protein family. Note, the wild-type (wt) and mutant (mut) sequence of FBE4 tested is larger than the given sequences (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#s4" target="_blank">Materials and Methods</a>). A positive and negative control RNA was included (not shown) and protein expression was confirmed by western blotting (not shown). (F) LAP-tagged FBF-2 associates with endogenous <i>gld-2</i> mRNA in RNA-coimmunoprecipitation experiments (RIPs) directed against the GFP portion of the fusion protein.</p