Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

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 p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 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

Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator

Stem cells are essential for tissue generation during the development of multicellular creatures, and for tissue homeostasis in adults. The great therapeutic promise of stem cells makes understanding their regulation a high priority. PUF RNA-binding proteins have a conserved role in promoting self-renewal of germline stem cells. Here we use a genome-wide approach to identify putative target mRNAs for the Caenorhabditis elegans PUF protein known as FBF. We find that putative FBF targets represent ∼7% of all protein-coding genes in C. elegans, implicating FBF as a broad-spectrum gene regulator. These putative FBF targets are enriched for regulators of meiotic entry and other components of the meiotic program as well as regulators of key developmental pathways. We suggest that these targets may be critical for FBF’s role in stem cell maintenance. Comparison of likely FBF target mRNAs with putative PUF target mRNAs from Drosophila and humans reveals 40 shared targets, including several established stem cell regulators. We speculate that shared PUF targets represent part of a broadly used module of stem cell control

Role of the Caenorhabditis elegans Multidrug Resistance Gene, mrp-4, in Gut Granule Differentiation

Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(−) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule

Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells

SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in <i>Caenorhabditis elegans</i>

<div><p>Central questions in regenerative biology include how stem cells are maintained and how they transition from self-renewal to differentiation. Germline stem cells (GSCs) in <i>Caeno-rhabditis elegans</i> provide a tractable <i>in vivo</i> model to address these questions. In this system, Notch signaling and PUF RNA binding proteins, FBF-1 and FBF-2 (collectively FBF), maintain a pool of GSCs in a naïve state. An open question has been how Notch signaling modulates FBF activity to promote stem cell self-renewal. Here we report that two Notch targets, SYGL-1 and LST-1, link niche signaling to FBF. We find that SYGL-1 and LST-1 proteins are cytoplasmic and normally restricted to the GSC pool region. Increasing the distribution of SYGL-1 expands the pool correspondingly, and vast overexpression of either SYGL-1 or LST-1 generates a germline tumor. Thus, SYGL-1 and LST-1 are each sufficient to drive “stemness” and their spatial restriction prevents tumor formation. Importantly, SYGL-1 and LST-1 can only drive tumor formation when FBF is present. Moreover, both proteins interact physically with FBF, and both are required to repress a signature FBF mRNA target. Together, our results support a model in which SYGL-1 and LST-1 form a repressive complex with FBF that is crucial for stem cell maintenance. We further propose that progression from a naïve stem cell state to a state primed for differentiation relies on loss of SYGL-1 and LST-1, which in turn relieves FBF target RNAs from repression. Broadly, our results provide new insights into the link between niche signaling and a downstream RNA regulatory network and how this circuitry governs the balance between self-renewal and differentiation.</p></div

SYGL-1 and LST-1 interact physically with FBF.

<p>(A) Yeast two hybrid assay. Full length SYGL-1 or LST-1 was fused to Gal4 activation domain (AD); PUF repeats of FBF-1(121–614) or FBF-2(121–632) were fused to LexA binding domain (BD). Interaction activates transcription of <i>HIS3</i> gene. (B and C) Yeast growth assays tested interaction between SYGL-1 and FBF (B) or LST-1 and FBF (C). Yeast strains were monitored for growth on synthetic defined media (SD), either lacking histidine or with histidine as a control. A <i>HIS3</i> competitive inhibitor (3-AT) improved stringency. (D) SYGL-1 and FBF-2 co-immunoprecipitation (IP). Western blots probed with α-FLAG to detect SYGL-1, α-V5 to detect FBF-2, and anti-α-tubulin as a loading control. 2% of input lysates and 20% of IP elutes were loaded. Exposure times of input and IP lanes are different, so band intensities are not comparable. RNA degradation by RNase A was confirmed. Genotypes for each lane: (1) <i>sygl-1(tm5040); qSi235[P</i>_<i>mex-5</i>::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (2) <i>sygl-1(tm5040); fbf-2(q931)[3xV5</i>::<i>fbf-2] qSi235[P</i>_<i>mex-5</i>::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (3) <i>sygl-1(tm5040); qSi297[P</i>_<i>mex-5</i>::<i>3xMYC</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (4) <i>sygl-1(tm5040); fbf-2(q932)[3xV5</i>::<i>fbf-2] qSi297[P</i>_<i>mex-5</i>::<i>3xMYC</i>::<i>sygl-1</i>::<i>tbb-2 3’end]</i>. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s007" target="_blank">S7 Fig</a></b> for data supporting functionality of epitope-tagged FBF-2. (E) Quantitative PCR of two signature FBF target mRNAs and a control mRNA after α-FLAG IP, using either <i>3xFLAG</i>::<i>sygl-1(ubiq)</i> for the experiment or <i>3xMYC</i>::<i>sygl-1</i>(<i>ubiq</i>) as the control. Abundance of mRNAs in input (gray bars) and IPs (blue bars) was calculated with the ΔΔ C_T method, using <i>rps-25</i> for normalization. Error bar indicates standard error. Asterisks indicate a statistically significant difference by 1-way ANOVA with Tukey HSD <i>post hoc</i> test. * p<0.05, ** p<0.01.</p

Models for stem cell pool regulation.

<p>(A) In each schematic, wild-type or manipulated extents of SYGL-1 (magenta) and LST-1 (orange) are shown above and GSC pool sizes are shown below. Wild type: GSC pool size corresponds to SYGL-1 rather than LST-1 extent; <i>sygl-1</i> mutant: pool size smaller than wild type and likely determined by smaller LST-1 extent; <i>lst-1</i> mutant: pool size not determined experimentally but likely similar to wild type, because progenitor zone is nearly the same size as normal; Extended SYGL-1 expression: moderate increase in SYGL-1 extent expands GSC pool (<i>tbb-2</i> 3’UTR transgene); Ubiquitous SYGL-1 expression: major expansion of SYGL-1 forms a massive tumor; Ubiquitous LST-1 expression: major expansion of LST-1 forms a massive tumor. (B) FBF forms a complex with SYGL-1 or LST-1 to repress differentiation RNAs. Red bars indicate repression; large pale blue circle represents an RNP granule. See text for explanation. (C) Loss of SYGL-1 and LST-1 triggers the switch from a naïve state to one primed-for-differentiation. See text for explanation.</p