DataSheet1_Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.DOC

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood.Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs.Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs.Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.</p

<i>Tryptophan hydroxylase</i> Is Required for Eye Melanogenesis in the Planarian <i>Schmidtea mediterranea</i>

<div><p>Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian <i>Schmidtea mediterranea</i>. We characterize the tryptophan hydroxylase (<i>tph</i>) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that <i>tph</i> is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in <i>S</i>. <i>mediterranea</i>, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in <i>tph</i> knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.</p></div

Pigment cup and photoreceptors remain intact in <i>tph(RNAi)</i> animals.

<p>(A-B) The planarian visual system. Photoreceptors (magenta) are visualized by immunofluorescence with VC-1 antibody against arrestin, and pigment cup cells (green) are visualized by fluorescent <i>in situ</i> hybridization of <i>tyrosinase (tyr)</i>. (A) depicts a maximum intensity confocal projection, whereas (B) represents a single confocal section. (C) By 10 days post-amputation, control and <i>tph(RNAi)</i> animals regenerate both photoreceptors and pigment cup cells, as indicated by VC-1 staining and <i>tyr</i> mRNA expression. Scale bars 20 μm.</p

<i>tph(RNAi)</i> animals regenerate pigment cup cells largely devoid of mature melanosomes.

<p>All micrographs are transverse sections of the planarian eye. (A) Electron micrograph of the photoreceptor rhabdomes and the pigment cup cells in control RNAi animals. Panel (B) is a magnified view of the pigment cup cells highlighting the mature melanosomes. (C-D) Electron micrograph of the photoreceptors of <i>tph</i> knockdown animals. In <i>tph</i> knockdowns the photoreceptors and the pigment cups are intact, but the melanosomes appear immature and less electron dense compared to controls. Scale bars in A and C are 2000 nm; in B and D are 500 nm.</p

<i>tph</i> is essential for photoreceptor pigmentation after regeneration.

<p>(A) Structure and genomic organization of the <i>tph</i> gene. Top, gene structure of <i>tph</i>; the 5’-untranslated region, the coding region, and the 3’-untranslated region are depicted in yellow, red and blue, respectively. Below, organization of genomic regions (supercontigs) that encode <i>tph</i>. (B-C) Whole-mount <i>in situ</i> hybridization to localize <i>tph</i> expression. The <i>tph</i> gene was expressed in the pigment cups, the peripharyngeal secretory cells (dorsal) and cells within the central and peripheral nervous systems (ventral). (D-E) RNAi-mediated knockdown of <i>tph</i>. In comparison to controls (D), <i>tph</i> knockdowns (E) regenerate pigment cups that appear to lack pigment; 21-day regenerates shown. (F-G) <i>in situ</i> hybridizations to detect <i>tph</i> mRNA levels following RNAi treatment. Relative to controls (F), <i>tph</i> dsRNA-treated animals show dramatically reduced <i>tph</i> mRNA expression; 21-day regenerates shown. Scale bars 200 μm.</p

Tryptophan derivatives rescue eye pigment in <i>tph</i> knockdowns.

<p>(A) Progress of pigment recovery after injection of 2.5 M 5-HTP is shown at hour-intervals. Eye pigment in <i>tph</i> knockdowns is largely recovered by 3 hr post-injection. (B) <i>tph(RNAi</i>) and control worms were injected with DMSO (n = 3), 100 mM tryptophan (n = 3), 100 mM 5-HTP (n = 5), 250 mM serotonin (n = 5), or 10 mM L-DOPA (n = 3). Images show phenotype at pre-injection and 24 hr time points. Insets highlight the increased pigmentation of control worms injected with 5-HTP vs. DMSO control. Scale bar is 200 μm. (C) Comparison of traditional and hypothesized melanin synthesis pathways.</p

<i>tph(RNAi)</i> animals lacking eye pigment are slower to orient to light.

<p>Orientation dynamics of control and <i>tph(RNAi</i>) planarians under low (A-C), medium (D-F) and high (G-I) light gradient conditions. Both control and <i>tph(RNAi)</i> worms respond to all three gradients by turning away from the light source, but <i>tph(RNAi)</i> worms react less efficiently, especially in low light gradients. (A, D, G) Center-of-mass tracking results for both control and tph(RNAi) populations (n = 10). Triangles indicate the orientation of the light gradient, with the light source on the right. All points are color-coded for time, as shown by color bar legends. At t = 0 s, the worms are manually oriented toward the light source. (B, E, H) Orientational order parameters as a function of time for both populations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127074#sec002" target="_blank">Methods</a>). The error bars show SEM at selected time points. The dashed y = 0 line is a guide for the eye showing the threshold between orientation biased toward the light souce (y > 0) and away from the light source (y < 0). Note the decrease in time scale as the gradient strength is increased, indicating a faster negative phototactic response of both populations at increased illumination. (C, F, I) Angular distributions of velocities at different time periods (corresponding to grayed regions in B, E and H) showing the reversal of polarity at different rates between control and <i>tph(RNAi</i>) worms. The size of the wedge indicates the proportion of worms traveling in the given direction in that time period, where 0° is towards the light source, and 180° is away from the light source. A two-way ANOVA test confirms statistical difference at the 1% level for TPH and control animals as well as for the three different gradient settings.</p