<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

Translational regulators maintain a robust proliferative zone in the adult germ line.

<p>(A) Expanded genetic circuitry of primarily translation regulators that fine-tunes the balance between proliferation and differentiation. A light grey box highlights pathway members that regulate differentiation onset. A dark grey box highlights redundant activities that promote GLD-1 expression, primarily when cells commit into meiotic progression (dashed line). See text for details. Note, this simplified circuitry focuses on the RNA regulatory network downstream of GLP-1/Notch and does neither include other known downstream RNA targets nor potential upstream protein regulators. (B) Diagram of translational control examples in proliferative germ cells. Next to <i>glp-1</i> mRNA, GLD-4 may also translationally activate additional mRNAs, encoding proliferation-promoting genes. Additional FBF-regulated mRNAs are known that promote the meiotic program. See text for details. (C) Diagram of translational control examples in differentiating germ cells. Additional GLD-regulated mRNAs are known that promote the proliferative fate. See text for details.</p

<i>gld-4</i> and <i>gls-1</i> promote onset of differentiation in parallel to <i>gld-2</i> and <i>nos-3</i>.

<p>(A) The current genetic wiring of the core regulatory network that regulates the balance between proliferation and differentiation onset. Two genetic pathways of redundantly acting translational regulators operate downstream of the translational repressor, FBF. A third, yet undefined, pathway has been evoked <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Fox1" target="_blank">[4]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Hansen1" target="_blank">[6]</a>. Note that not all genes are equivalent in the two pathways; only <i>gld-3 nos-3</i> double mutants lack any signs of differentiation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Eckmann1" target="_blank">[7]</a>. (B–H) Complete gonads stained with DAPI (left column), and with α-REC-8 and α-HIM-3 (right column) antibodies. Dashed boxes in B and C are close ups of D and E. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen-1004647-t001" target="_blank">Table 1</a> for the total number of analyzed germ lines. (D–H) Distal region of extruded gonads. Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. Scale bars: 50 µm.</p

<i>gld-4</i> and <i>gls-1</i> promote onset of differentiation in parallel to <i>gld-2</i> and <i>gld-1</i>.

<p>(A–F) Distal region of extruded gonads stained with DAPI, and with α-REC-8, α-HIM-3 (A–E), and α-pSUN-1 (F) antibodies. Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. Scale bars: 50 µm. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen-1004647-t001" target="_blank">Table 1</a> for the total number of analyzed germ lines. (E–F) <i>gld-1 gls-1</i> double mutant germ lines possess a mitosis-to-meiosis boundary, albeit HIM-3 fails to be detected in E. A strong reduction of nucleoplasmic REC-8, the appearance of crescent-shaped nuclei in meiotic prophase (circles in E), and the abundant expression of pSUN-1 (F) reveal onset of differentiation.</p