FBF Binds Specifically to FBEs in <i>mpk-1</i> 3′UTR

<div><p>(A) Two predicted FBF binding elements in <i>mpk-1</i> 3′UTR.</p><p>(B) Schematic of yeast three-hybrid assay. Briefly, a hybrid RNA carrying the query sequence can bridge the LexA-MS2 and GAL4AD-FBF hybrid proteins if FBF binds, but it cannot bridge them if FBF fails to bind.</p><p>(C) Nucleotide sequences of predicted FBEs, aligned in register with their conserved UGURHHAU motifs (bold in gray boxes). Each wild-type sequence is followed by its mutant (*), in which UGU is replaced by aca (mutated nucleotides are lowercase). Controls included the <i>fem</i>-3 FBE in the <i>fem-3</i> 3′UTR, previously called the PME [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b012" target="_blank">12</a>], which served as a positive control for FBF binding, and the <i>hb (hunchback)</i> NRE, which served as a negative control for FBF binding and a positive control for PUF-8 binding [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b055" target="_blank">55</a>].</p><p>(D) Three-hybrid interactions assayed by β-galactosidase activity. Nomenclature and conventions are the same as in (C). Standard deviation bars were calculated from three independent experiments.</p><p>(E) Purified FBF-2 binds <i>mpk-1</i> FBEa and <i>mpk-1</i> FBEb in gel mobility assays, but not to mutants (*) with an altered consensus as detailed in (C). Apparent affinities of MPK-1 FBEa and FBEb are 93 nM and 320 nM, respectively.</p><p>(F) Coimmunoprecipitation of <i>mpk-1</i> mRNA with an epitope-tagged FBF. <i>eft-3</i> served as a negative control, and <i>gld-1</i> served as a positive control [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b015" target="_blank">15</a>].</p><p>(G) Sequence alignment of <i>mpk-1</i> FBEs from C. elegans, <i>C. briggsae,</i> and C. remanei.</p></div

<i>mpk-1</i> Expression in the C. elegans Germline

<div><p>(A) Schematics of <i>mpk-1a</i> and <i>mpk-1b</i> mRNAs. Box, exon; connecting line, intron; ATG, initiation codon; TAG, termination codon. Below schematics: thick bars, extent of probes used for in situ hybridization; arrows, primer pairs used for RT-PCR.</p><p>(B) Semiquantitative RT-PCR of RNA prepared from adult hermaphrodites that either had an essentially normal germline [<i>glp-1(q224)</i> grown at 15 °C], or had virtually no germline [<i>glp-1(q224)</i> grown at 25 °C] (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#s4" target="_blank">Materials and Methods</a>). <i>unc-54</i> was used as a control.</p><p>(C) Western blot. MPK-1A protein is ≈45 kDa, MPK-1B is ≈55 kDa, and α-TUB is α-tubulin. Proteins were extracted from adult hermaphrodites that were either wild-type (wt), <i>glp-1(q224)</i> grown at 25 °C (GL−), or <i>mpk-1(ga117)</i> putative null homozygotes <i>[mpk-1(0)]</i>.</p><p>(D–F) In situ analysis of dissected adult hermaphrodite germlines. (D) Total <i>mpk-1</i> RNA was assessed using the <i>mpk-1ab</i> antisense probe shown in (A). (E) <i>mpk-1b</i> RNA was assessed using an isoform-specific antisense probe shown in (A). (F) Negative control, using an <i>mpk-1b</i>−specific sense probe.</p><p>(G–L) Immunocytochemistry of dissected adult hermaphrodite germlines. All were stained using both MPK-1 antibodies (G, I, K) and DAPI (H, J, L). Distal end, arrowhead; dotted lines, boundaries between regions of germline maturation [MR (mitotic region), TZ (transition zone), PR (pachytene region), OO (oocytes), SP (sperm)]; PEX (pachytene exit defect). (G, H) Same wild-type germline. (I, J) Same <i>mpk-1(0)</i> germline. (K, L) Same <i>mpk-1b(RNAi)</i> germline.</p></div

FBF Represses <i>mpk-1b</i> Germline Expression

<div><p>(A) Western blot analysis. MPK-1B abundance increases when FBF is removed. Loading control, α-tubulin. Band intensity was measured using ImageJ software.</p><p>(B–E) Dissected adult hermaphrodite germlines with FBF (B, C) and without FBF (D, E); genotypes noted in images. Distal end, arrowhead; dotted lines, boundaries between regions of germline maturation (same conventions as detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-g001" target="_blank">Figure 1</a>G–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-g001" target="_blank">1</a>L legend). (B) MPK-1 staining in tumorous germlines that contain FBF. (C) DAPI staining of germline shown in (B). (D) MPK-1 staining in tumorous germlines that have no FBF. (E) DAPI staining of germline shown in (D). Germlines were treated identically, and images were taken with the same settings at the same magnification for comparison.</p><p>(F) Quantitation of MPK-1 protein in Tum+FBF (<i>gld-1</i>, <i>n</i> = 7), Tum−FBF (<i>gld-1; fbf-1 fbf-2</i>, <i>n</i> = 5), and <i>mpk-1(0)</i> (<i>n</i> = 3) mutants. The intensity of MPK-1 protein was quantified using ImageJ software. The <i>x</i>-axis represents distance from distal tip of the germline, and the <i>y</i>-axis is pixel intensity. Dotted lines show boundaries of transition zone in Tum+FBF (<i>gld-1</i>) mutants; Tum−FBF (<i>gld-1; fbf-1 fbf-2)</i> mutants do not have a transition zone.</p></div

Regulation of MAPK Activity by PUF and MKPs

<div><p>(A) Conserved positive and negative regulators of MAPK expression and activity. See text for further explanation.</p><p>(B) MAPK regulation in the C. elegans germline. The distal end of the germline is controlled by Notch signaling from the distal tip cell (DTC), which provides the stem cell niche [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b011" target="_blank">11</a>]. FBF/PUF RNA-binding proteins are present in the distalmost germ cells, which include stem cells. FBF maintains germ cells in a naïve and undifferentiated state, in part by repression of <i>mpk-1</i> expression (present work). In addition, FBF represses <i>lip-1/MKP</i> mRNA in the stem cell region [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b023" target="_blank">23</a>]; more proximally, where FBF abundance decreases, LIP-1/MKP inhibits MPK-1/MAPK activity; yet more proximally, LET-60/RAS activates MPK-1/MAPK to promote oocyte differentiation and apoptosis. OO, oocyte.</p></div

Conservation of PUF Binding to Regulatory Elements in Human Erk2 and p38α 3′UTRs

<div><p>(A) Putative PUM2 binding elements (NREs) in Erk2 and p38α 3′UTRs; filled triangles, elements that bound in vitro; empty triangles, elements that did not bind in vitro.</p><p>(B) Nucleotide sequence of predicted NREs. Sequences are aligned in register with their conserved UGUANAU motif (bold in gray boxes). Mutated nucleotides are lowercase.</p><p>(C) Three-hybrid interactions assayed by β-galactosidase activity. Standard deviation bars were calculated from three independent experiments.</p><p>(D) Purified PUM2 binds Erk2 NRE as well as p38α NREa and NREb in gel mobility assays, but does not bind mutants (*) with an altered consensus as detailed in (B).</p><p>(E) Sequence alignment of Erk2 NREs from human and mouse.</p></div

Differentially expressed isoforms (as predicted by LongSAGE tag positions) for the transcript (see text)

The tag sequence at position 9 results in the loss of the 3' UTR region targeted by evolutionarily conserved miRNAs. Putative miRNA target sites were predicted using miRanda [34] and are represented by hashed boxes.<p><b>Copyright information:</b></p><p>Taken from "LongSAGE profiling of nine human embryonic stem cell lines"</p><p>http://genomebiology.com/2007/8/6/R113</p><p>Genome Biology 2007;8(6):R113-R113.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC2394759.</p><p></p

Expression of selected transcripts during embryoid body differentiation

qPCR was used to monitor expression of selected transcripts in ESCs stimulated to differentiate into embryoid bodies. Three control markers, Oct4, Lin28 and Msx1, were included. Expression levels are reported as the mean of triplicate measurements and are normalized to GAPDH.<p><b>Copyright information:</b></p><p>Taken from "LongSAGE profiling of nine human embryonic stem cell lines"</p><p>http://genomebiology.com/2007/8/6/R113</p><p>Genome Biology 2007;8(6):R113-R113.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC2394759.</p><p></p