Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3′ Untranslated Region

Translation termination at premature termination codons (PTCs) triggers degradation of the aberrant mRNA, but the mechanism by which a termination event is defined as premature is still unclear. Here we show that the physical distance between the termination codon and the poly(A)-binding protein PABPC1 is a crucial determinant for PTC recognition in human cells. “Normal” termination codons can trigger nonsense-mediated mRNA decay (NMD) when this distance is extended; and vice versa, NMD can be suppressed by folding the poly(A) tail into proximity of a PTC or by tethering of PABPC1 nearby a PTC, indicating an evolutionarily conserved function of PABPC1 in promoting correct translation termination and antagonizing activation of NMD. Most importantly, our results demonstrate that spatial rearrangements of the 3′ untranslated region can modulate the NMD pathway and thereby provide a novel mechanism for posttranscriptional gene regulation

A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice

The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration

Combined deletion of Lgr4 and Lgr5 impairs embryonic mouse development

Lgr4 and Lgr5 proteins are known markers of adult and embryonic tissue stem cells in various organs. However, the role of these proteins in propagating and maintaining individual tissue stem cell compartments is still controversial. While it was reported that Lgr4 is dispensable for normal embryonic gut development, Lgr4 deletion functionally impaired maintenance of the postnatal and adult intestinal crypt stem cell compartment. Furthermore, concomitant deletion of Lgr4 in Lgr5-null embryos was able to rescue their perinatal lethality, whereas combined deletion of Lgr4 and Lgr5 in adult mice exacerbated the latter phenotype, suggesting antagonistic or complementary functions of both receptors, respectively. While the effects of Lgr4 deletion during embryonic skin and kidney development have been reported, combined deletion of Lgr4 and Lgr5 has not been studied to date. To elucidate the functions of Lgr4 and Lgr5 during intestinal crypt development and to study their role in developing kidney and skin, we generated homozygous mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). Lgr4 deletion resulted in loss of Lgr5+ intestinal stem cells and impaired proliferation in the developing gut of E16.5 mice, a phenotype that was not further increased nor ameliorated by combined deletion of Lgr4 and Lgr5 (Lgr4/5dKO). In skin, E16.5 Lgr4KO and Lgr4/5dKO mice displayed impaired proliferation of basal cell progenitors accompanied by reduced epidermal thickness and reduced numbers of hair follicles. In contrast to E16.5 Lgr4KO mice, Lgr4/5dkO mice did neither show dilated kidney tubules nor cysts. However, E16.5 Lgr4/5dKO mice showed impaired kidney cell proliferation which was not observed in Lgr4KO mice. In summary, our data show that combined deletion of Lgr4 and Lgr5 impairs embryonic development with a dominant role of Lgr4 and support a complementary rather than an antagonistic function for both receptors

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis

Background: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. Methodology: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. Principal Findings: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. Significance: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis

Protection of inducible Sgpl1-deficient mice in EAE.

<p>Tamoxifen-induced Sgpl1^Flox/Flox Cre^+/−, Sgpl1^Flox/Flox Cre^−/−, Cre^+/−, and Cre^−/− mice (n = 6–10/group) were immunized with MOG emulsified in Complete Freund’s Adjuvans. Data from one representative experiment out of three independent studies are shown. <i>A</i>, Incidence of mice with a clinical EAE score ≥1; <i>B</i>, clinical score; <i>C</i>, body weight. For histological analysis thoracic sections of spinal cord tissue from Sgpl1^Flox/Flox Cre^+/− and Sgpl1^Flox/Flox Cre^−/− mice undergoing EAE (day 24) were stained (<i>D</i>) with H&E to visualize CNS-invading cells (scale bar is 500 µm, arrows highlight areas of inflammation); <i>E</i>, for CD3⁺ T cells (scale bar is 500 µm, rectangles indicate area of magnification, where scale bar represents 100 µm); and <i>F</i>, with solochrome to assess the integrity of the myelin sheath (scale bar is 500 µm, arrows highlight areas of beginning demyeliniation).</p

Generation of the modRosa26^FRT locus and a reporter strain for strong EGFP expression.

<p>(A) Flp-RMCE into the modRosa26^FRT locus, replacing the HygR selection cassette in the modRosa26^FRT ES cells with an FRT/FRTwt-flanked sequence in the Flp-RMCE targeting plasmid. The Flp-RMCE targeting plasmid contains the pCAG promoter followed by a LoxP-flanked (floxed) STOP cassette, the EGFP cDNA and a NeoR cassette, flanked as a group by FRT/FRTwt sites. Successfully targeted ES cells were used to generate mR26-CS-EGFP mice. After crossing mR26-CS-EGFP mice with several Cre mice, ubiquitous or tissue-restricted EGFP reporter expression could be obtained. (B) mR26CS-EGFP/CMV-Cre E12.5 embryos show ubiquitous EGFP expression, while mR26CS-EGFP/Nestin-Cre mice show EGFP expression restricted to the brain and neural tube. EGFP expression in E10.5 mR26CS-EGFP/Myf5-Cre embryos was restricted to the somites (magnified inset), limbs and parts of the brain. (C) Neural stem cells isolated from E14.5 mR26CS-EGFP/Nestin-Cre mice form neurospheres with ubiquitous and strong EGFP fluorescence, while mR26CS-EGFP mice show no fluorescence. These cells were subsequently differentiated (lower panels), showing strong EGFP fluorescence in mR26CS-EGFP/Nestin-Cre-derived cells (counterstained with DAPI, the neuronal marker Tuj1 and the glial marker GFAP). (D) Adult mR26CS-EGFP/Glast-CreERT2 mice show strong and specific EGFP fluorescence in astrocytes and in the adult neural stem cell niche (asterisks) upon tamoxifen administration. EGFP+ adult neural stem cells are present in the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ). Recombined EGFP+ cells from the SVZ can be traced through the rostral migratory stream into the olfactory bulb. NeuN stains for mature neurons (blue) and BLBP for adult neural stem cells and astrocytes (red). Note: no EGFP-antibody staining was used in C–E, since the mR26CS-EGFP reporter mouse offers very high EGFP expression levels which can be easily detected without any staining.</p

Generation of luciferase reporter mice (mR26CS-Luc).

<p>(A) Flp-RMCE into the modRosa26^FRT locus was performed, replacing the HygR in the modRosa26^FRT ES cells with the FRT/FRTwt-flanked sequence from the Flp-RMCE targeting plasmid. The Flp-RMCE targeting plasmid contains the pCAG promoter followed by a LoxP-flanked (floxed) STOP cassette (STOP2), the luciferase cDNA and a NeoR cassette, flanked as a group by FRT3/FRTwt sites. Successfully targeted ES cells were used to generate mR26CS-Luc mice. After crossing mR26CS-Luc mice with Cre mice, ubiquitous or tissue-restricted luciferase reporter expression can be obtained to monitor reporter expression in living mice upon Luciferin injection. Using Xenogen imaging, adult mR26CS-Luc/CMV-Cre mice show ubiquitous luciferase activity throughout the body (B), while mR26CS-Luc/AlbCre mice show luciferase activity restricted to the liver upon Luciferin injection (C). Luciferin-injected mR26SC-Luc control mice never showed luciferase activity.</p

Sphingolipid concentration in selected tissues of inducible Sgpl1-deficient mice.

<p>Two weeks after tamoxifen induction, tissues of Sgpl1^Flox/Flox Cre^+/− mice (open bars) and of Sgpl1^Flox/Flox Cre^−/− controls (filled bars) were obtained (n = 5/group). Tissues were extracted and sphingolipids were quantified by LC/MS. <i>A, B,</i> S1P; <i>C, D</i>, Sph; <i>E,</i> C16-ceramide. <i>A, C,</i> and <i>E</i> show absolute concentrations per weight of tissue; <i>B and D</i> show fold increase in inducible KO mice.</p