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. 2007 Dec 28;3(12):e233.
doi: 10.1371/journal.pgen.0030233.

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Affiliations

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Myon-Hee Lee et al. PLoS Genet. .

Abstract

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3' untranslated region (3' UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3'UTR elements in both Erk2 and p38alpha mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38alpha 3' UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

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Conflict of interest statement

Competing interests. JAT owns stock, serves on the Board of Directors, and serves as Chief Scientific Officer of Cellular Dynamics International and Stem Cell Products. JAT also serves as Scientific Director of the WiCell Research Institute.

Figures

Figure 1
Figure 1. mpk-1 Expression in the C. elegans Germline
(A) Schematics of mpk-1a and mpk-1b 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. (B) Semiquantitative RT-PCR of RNA prepared from adult hermaphrodites that either had an essentially normal germline [glp-1(q224) grown at 15 °C], or had virtually no germline [glp-1(q224) grown at 25 °C] (see Materials and Methods). unc-54 was used as a control. (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), glp-1(q224) grown at 25 °C (GL−), or mpk-1(ga117) putative null homozygotes [mpk-1(0)]. (D–F) In situ analysis of dissected adult hermaphrodite germlines. (D) Total mpk-1 RNA was assessed using the mpk-1ab antisense probe shown in (A). (E) mpk-1b RNA was assessed using an isoform-specific antisense probe shown in (A). (F) Negative control, using an mpk-1b−specific sense probe. (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 mpk-1(0) germline. (K, L) Same mpk-1b(RNAi) germline.
Figure 2
Figure 2. FBF Represses mpk-1b Germline Expression
(A) Western blot analysis. MPK-1B abundance increases when FBF is removed. Loading control, α-tubulin. Band intensity was measured using ImageJ software. (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 Figure 1G–1L 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. (F) Quantitation of MPK-1 protein in Tum+FBF (gld-1, n = 7), Tum−FBF (gld-1; fbf-1 fbf-2, n = 5), and mpk-1(0) (n = 3) mutants. The intensity of MPK-1 protein was quantified using ImageJ software. The x-axis represents distance from distal tip of the germline, and the y-axis is pixel intensity. Dotted lines show boundaries of transition zone in Tum+FBF (gld-1) mutants; Tum−FBF (gld-1; fbf-1 fbf-2) mutants do not have a transition zone.
Figure 3
Figure 3. FBF Binds Specifically to FBEs in mpk-1 3′UTR
(A) Two predicted FBF binding elements in mpk-1 3′UTR. (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. (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 fem-3 FBE in the fem-3 3′UTR, previously called the PME [12], which served as a positive control for FBF binding, and the hb (hunchback) NRE, which served as a negative control for FBF binding and a positive control for PUF-8 binding [55]. (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. (E) Purified FBF-2 binds mpk-1 FBEa and mpk-1 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. (F) Coimmunoprecipitation of mpk-1 mRNA with an epitope-tagged FBF. eft-3 served as a negative control, and gld-1 served as a positive control [15]. (G) Sequence alignment of mpk-1 FBEs from C. elegans, C. briggsae, and C. remanei.
Figure 4
Figure 4. FBF and LIP-1 Function Redundantly to Control Distribution of Activated MPK-1 and to Protect Germ Cells From Apoptosis
(A–D) Dissected adult germlines stained with α-DP-MAPK antibody, which is specific for activated MPK-1. Germlines were treated identically, and images were taken with the same settings at the same magnification. Conventions are same as described in Figure 1. (E–J) Nomarski (left column) and SYTO 12 staining (right column) of adult hermaphrodite germlines (24 h after L4 at 20 °C or 18 h past L4 at 25 °C). The vital dye SYTO 12 stains apoptotic germ cells (arrowheads in F, H, and J). (K) Average number of apoptotic germ cells per gonad arm. Standard deviation bars were calculated from three independent experiments.
Figure 5
Figure 5. Conservation of PUF Binding to Regulatory Elements in Human Erk2 and p38α 3′UTRs
(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. (B) Nucleotide sequence of predicted NREs. Sequences are aligned in register with their conserved UGUANAU motif (bold in gray boxes). Mutated nucleotides are lowercase. (C) Three-hybrid interactions assayed by β-galactosidase activity. Standard deviation bars were calculated from three independent experiments. (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). (E) Sequence alignment of Erk2 NREs from human and mouse.
Figure 6
Figure 6. The Erk2 and p38α 3′UTRs Control Reporter Expression in hESC
(A) Schematic diagram of DNA constructs used for reporter assays. EGFP reporters all use the same EF1p promoter and contain one of the following 3′UTRs: Erk2 or p38α wild-type (wt) or Erk2 or p38α mutant (mut). The Pum2::FLAG construct contained PUM2 protein coding region plus three copies of FLAG sequences. All transfections included a luciferase-expressing plasmid to control for transfection efficiency (see Materials and Methods). (B–I, K–N) Photomicrographs of hESCs; experimental plasmids are noted in images. All steps were performed identically to compare EGFP expression levels; images shown were representative of at least three independent experiments. (J, O) Western blot analysis. PUM2 represses expression of EGFP reporter constructs bearing either Erk2 or p38α 3′UTRs. The intensities were normalized using β-actin to control for cell number and luciferase (LUC) to control for transfection efficiency.
Figure 7
Figure 7. Regulation of MAPK Activity by PUF and MKPs
(A) Conserved positive and negative regulators of MAPK expression and activity. See text for further explanation. (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 [11]. 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 mpk-1 expression (present work). In addition, FBF represses lip-1/MKP mRNA in the stem cell region [23]; 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.

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References

    1. Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev. 2004;68:320–344. - PMC - PubMed
    1. Lackner MR, Kornfeld K, Miller LM, Horvitz HR, Kim SK. A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans . Genes Dev. 1994;8:160–173. - PubMed
    1. Wu Y, Han M. Suppression of activated Let-60 Ras protein defines a role of Caenorhabditis elegans Sur-1 MAP kinase in vulval differentiation. Genes Dev. 1994;8:147–159. - PubMed
    1. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994;4:82–89. - PubMed
    1. Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J. 2000;14:6–16. - PubMed

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