Aronia meranocalpa), also called black chokeberry, contains abundant anthocyanins that have received considerable interest for their possible relations to vascular health.Inflammation in endothelial cells induces production of inflammatory cytokines and monocytes adhesion, which are crucial events in the initiation of atherosclerosis. Aronia berry  pretreated with aronia berry extract were stimulated with tumor necrosis factor-alpha (TNF-α). The expression levels of cytokines and adhesion molecules were analyzed. To investigate the effects of aronia berry extract on the adhesion of THP-1 monocytic cell, the static adhesion assay was carried out. The possible molecular mechanisms by which aronia berry extract regulated vascular inflammatory responses were explored.The mRNA expressions of interleukins  and monocyte chemoattractant protein-1 (MCP-1) upregulated by TNF-α were significantly suppressed by pretreatment with aronia berry extract. Aronia berry extract decreased TNF-α-induced monocyte/endothelial adhesion and suppressed vascular cell adhesion molecule-1 (VCAM-1) expression, but did not affect intercellular adhesion molecule-1 (ICAM-1) expression. Moreover, aronia berry extract decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the nuclear levels of STAT3 and interferon regulatory transcription factor-1 (IRF1). The nuclear translocation of nuclear factor-kappa B (NF-κB) was not inhibited by aronia berry extract.Aronia berry extract could exert anti-atherosclerotic effects on TNF-α-induced inflammation through inhibition of STAT3/IRF1 pathway in vascular endothelial cells. Aronia berry contains abundant anthocyanins and recently its functionality attracts attention.The effect of aronia berry extract on vascular endothelial inflammation was investigated in the cultured human endothelial cells stimulated with TNF-α.Aronia berry extract attenuated the expressions of inflammatory cytokines and cell adhesion molecules through the inhibition of STAT3/IRF1 pathway.Anti-inflammatory effect of aronia berry extract may be useful to reduce risk factors for atherosclerosis.Vascular endothelial inflammation is well known to be an initial step in atherosclerosis , 2. VascRegarding the mechanisms underlying the regulation of the expression of inflammatory cytokines and adhesion molecules, the involvement of the nuclear factor-kappa B (NF-κB) pathway and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is well known . These pAronia meranocalpa), known as black chokeberry, is a shrub native to North America and is commonly consumed as juice, wine, and jam  under stimulation with TNF-α as a model of vascular endothelial inflammation.In the present study, we explored the effects of aronia berry extract on inflammation in vascular endothelial cells, which are closely involved in the progression of arteriosclerosis. We conducted A concentrated ethanol extract of aronia berry was provided by POLA Chemical Industries Inc. . According to a high-performance liquid chromatography (HPLC) analysis conducted by the manufacturer, the total anthocyanins content of the aronia berry extract powder was 36.49%, which consisted of 23.52% cyanidin-3-O-galactoside, 10.48% cyanidin-3-O-arabinoside, 1.36% cyanidin-3-O-xyloside, and 1.13% cyanidin-3-O-glucoside. The powder extract was dissolved in deionized water at 5 mg/mL and used in the experiments. RPMI-1640 medium, Hank’s balanced salts solution (HBSS), and 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were purchased from Sigma-Aldrich . Fetal bovine serum (FBS), penicillin/streptomycin, novoheparin, and recombinant human fibroblast growth factor (hFGF) basic were obtained from Biowest , GIBCO , Mochida Pharmaceutical (Tokyo), and R&D Systems , respectively.2. HUVECs were pretreated with aronia berry extract (0–25 μg/mL) and then stimulated with TNF-α (10 ng/mL). The concentration of aronia berry extract added to the cells was set within the range of noncytotoxic based on the results of MTT assay (data not shown).HUVECs were obtained from Lonza , grown in RPMI-1640 medium supplemented with 20% FBS, 1% penicillin/streptomycin, 0.5% novoheparin, and 0.05% recombinant hFGF basic at 37°C and 5% COIL1B), forward 5’-CTG TAC GAT CAC TGA ACT GC-3’ and reverse 5’-CAC CAC TTG TTG CTC CAT ATC-3’; for IL-6 (IL6), forward 5’-GGT ACA TCC TCG ACG GCA TC-3’ and reverse 5’-GCC TCT TTG CTG CTT TCA CAC-3’; for gp130 (IL6ST), forward 5’-CTG GGA GTG CTG TTC TGC TTT-3’ and reverse 5’-GCC TTG GAG GAG TGT GAG GT-3’; for IL-8 (CXCL8), forward 5’-AAA CTG GGT GCA GAG GGT TG-3’ and reverse 5’-TGG CAT CTT CAC TGA TTC TTG G-3’; for MCP-1 (CCL2), forward 5’-ATC ACC AGC AGC AAG TGT CC-3’ and reverse 5’-CAA GTC TTC GGA GTT TGG GTT T-3’; for VCAM-1 (VCAM1), forward 5’-CAT GGA ATT CGA ACC CAA ACA-3’ and reverse 5’-GAC CAA GAC GGT TGT ATC TCT GG-3’; and for ICAM-1 (ICAM1), forward 5’-GGG CAG TCA ACA GCT AAA ACC TT-3’ and reverse 5’-CAC CTG GCA GCG TAG GGT AA-3’.RNA was extracted from cells with the total RNA extract reagent RNAiso Plus . We synthesized complementary DNAs from 2 μg of the total RNA by using a High Capacity cDNA Reverse Transcription Kit . A real-time polymerase chain reaction (PCR) was performed on an ABI 7300 cycler  with Power SYBR Green PCR mix (Thermo Fisher Scientific). We used r18s rRNA as the endogenous control. For all samples, the quantity was determined from the relative standard curve and normalized with an endogenous control. The primer sequences are as follows: for r18s, forward 5’-ACT CAA CAC GGG AAA CCT CAC-3’ and reverse 5’-CAG ACA AAT CGC TCC ACC AA-3’; for IL-1β  containing protease inhibitor cocktail . Aliquots of protein were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and transferred to an Immobilon-P membrane . After blocking, the membranes were incubated with primary antibodies: anti-gp130, anti-NF-κB, anti-VCAM-1, anti-ICAM-1, anti-LaminB , anti-stat3, anti-phospho-stat3, anti-IRF-1, anti-β-actin, and anti-GAPDH . Each bound antibody was then detected with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody. Chemiluminescent detection of specific proteins was developed with the use of ECL Select Western Blotting Detection Reagent . All signals were captured by the LAS-4000 system .We obtained the human monocytic cell line THP-1 from the RIKEN Cell Bank . THP-1 cells were incubated with the fluorescent dye BCECF for 20 min at 37°C. The fluorescently labeled THP-1 cells were added to the HUVECs and incubated for10 min. The unbound THP-1 cells were washed away, and fluorescent images were obtained with a fluorescence microscope. The fluorescent intensity of the adherent THP-1 cells was also measured at Ex.482 nm/Em.528 nm by a PowerScan-HT .P values < 0.05 were accepted as significant. All results were analyzed with the GraphPad Prism 5 software package .Values are presented as the mean ± SD. We performed a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to compare treatment groups. IL1B and IL6 by the addition of TNF-α was significantly suppressed by aronia berry extract. Moreover, IL6ST (gp130), a signaling molecule of IL-6, was suppressed by aronia berry extract at both the mRNA and protein levels (CXCL8 (IL-8) and CCL2 (MCP-1)  .CAMs are greatly involved in plaque formation by mediating leukocyte migration. Here we observed that aronia berry extract concentration-dependently reduced TNF-α-stimulated VCAM-1 induction at both the mRNA and protein levels . HoweverTo investigate the effect of aronia berry extract on monocyte-endothelial interactions, we performed an adhesion assay under static (no shear stress) conditions. The stimulation with TNF-α significantly increased the adhesion of THP-1 cells to HUVECs. In contrast, pretreatment with aronia berry extract (≥12.5 μg/mL) significantly reduced the adherent cells to HUVECs .Inflammatory molecules are known to be regulated by the nuclear transcription factors NF-κB and STAT3. To investigate the mechanism underlying the anti-inflammatory effects of aronia berry extract, we examined the nuclear translocation of NF-κB and STAT3. The aronia berry extract inhibited the TNF-α-induced STAT3 phosphorylation and suppressed the nuclear expression , but it The results of our present experiments revealed that aronia berry extract attenuated TNF-α-induced proinflammatory changes in endothelial cells, which are involved in the upregulation of inflammatory cytokines and chemokines  and an adhesion molecule (VCAM-1) and an increase in endothelial-monocyte adhesion. Aronia berry extract also decreased the expression of gp130, a signaling molecule of IL-6. Interestingly, aronia berry extract downregulated the TNF-α-induced VCAM-1 expression but not ICAM-1 expression, and it decreased the nuclear expressions of STAT3 and IRF1. Our data indicate that the anti-inflammatory effects of aronia berry extract are exerted, at least in part, through a suppression of STAT3 activation.An overexpression of inflammatory cytokines/chemokines, such as IL-1β, -6, -8, and MCP-1, induces cell migration and adhesion, angiogenesis, and vascular permeability to exacerbate atherosclerosis –25. In tAronia berry extract contains a rich amount of anthocyanins, mainly cyanidin-3-O-galactoside and cyanidin-3-O-arabinoside, which we suspect contributed to our present findings. Earlier studies have demonstrated that several anthocyanins or anthocyanin-rich plant extracts inhibited the expression of inflammatory cytokines/chemokines and adhesion molecules and the adhesion of monocytes to vascular endothelial cells –31. As w13C-labeledcyanidin-3-glucoside, on TNF-α-induced VCAM-1 and IL-6 production. They found that the greatest inhibition of VCAM-1 was observed in response to the 24 h metabolite signature, suggesting the metabolites of lower intestinal microbial origin are responsible for anti-inflammatory effects  was suppressed by aronia berry extract. The aronia berry extract used in this study at 25 μg/mL contained approximately 20 μmol/L of cyanidin glycosides. Similar concentrations were used in the studies of anthocyanins (1–100 μmol/L) , 32, 33. effects . It shouin vivo efficacy of aronia berry extract are needed.In conclusion, we observed that aronia berry extract could regulate the expressions of inflammatory cytokines and cell adhesion molecules through the inhibition of STAT3 activation in TNF-α-induced vascular endothelial cells. Our results indicate that aronia berry extract may be effective in the reduction of risk factors associated with atherosclerosis, but further investigations of the detailed molecular mechanisms and the This work was partly supported by JSPS KAKENHI  from the Japan Society for the Promotion of Science. The authors declare no potential conflicts of interest.


C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity.microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during  hrpk-1 role in development and its functional interactions with multiple miRNA families. We suggest that HRPK-1 promotes miRNA activity on multiple levels in part by contributing to miRNA processing and by coordinating with miRISC at the level of target RNAs. This work contributes to our understanding of how RNA binding proteins and auxiliary miRNA cofactors may interface with miRNA pathways to modulate miRNA gene regulatory activity.microRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level. The core microRNA Induced Silencing Complex (miRISC), composed of Argonaute, mature microRNA, and GW182 protein effector, assembles on the target messenger RNA and inhibits translation or leads to messenger RNA degradation. RNA binding proteins interface with miRNA pathways on multiple levels to coordinate gene expression regulation. Here, we report identification and characterization of HRPK-1, a conserved RNA binding protein, as a physical and functional interactor of miRNAs. We confirm the physical interaction between HRPK-1, an hnRNPK homolog, and Argonaute ALG-1. We report characterizations of  Robust regulation of gene expression is essential for normal development and cellular homeostasis. microRNAs (miRNAs), small non-coding RNAs ~22nt in length, negatively regulate gene expression at the post-transcriptional level. miRNAs can act as developmental switches or can fine tune the expression of the target genes . Plet-7 family of miRNAs , . I. Ilet-7 rgonaute , hypotheK homology . F. FK homo protein , 22]. I. IK homo protein , althougpression .PLK1 PLK1  animals have increased defects compared to hrpk-1(zen17) [m+z-] in sterility  allele is weakly semi-dominant and antimorphic in nature (hrpk-1(tm5522) mutants almost always exhibit more severe phenotypes than hrpk-1(zen17) null animals (hrpk-1(tm5522)/+ [m-z+] animals carrying only a single copy of the wild type allele exhibited higher fertility defects and vulval bursting than hrpk-1(zen17)/+ [m-z+] animals /+ [m+z+] animals show a modest reduction in brood size (hrpk-1(tm5522) allele (hrpk-1(tm5522)⚥/hrpk-1(zen17)♂ produced by hrpk-1(tm5522) mothers and hrpk-1(zen17) fathers) exhibit higher rates of sterility (hrpk-1(zen17) [m-z-] animals lacking functional hrpk-1 completely. Animals carrying a single copy of maternally provided hrpk-1(tm5522) allele (hrpk-1(tm5522)⚥/hrpk-1(zen17)♂also show more severe phenotypes than hrpk-1(tm5522)♂/ hrpk-1(zen17)⚥ animals produced by hrpk-1(tm5522) fathers and hrpk-1(zen17) mothers  allele.Our genetic analysis suggests that n nature . Firstly animals . Secondlood size  and embrood size  and vulvood size  not normterility , have smterility , and higterility  than hrp mothers , likely hrpk-1 functions in miRNA-dependent cell fate specification, we examined potential genetic interactions between hrpk-1 and let-7 family of miRNAs that control temporal cell fate specification throughout larval development. The let-7 miRNAs regulate the stage specific cell gene expression programs in a number of tissues, with the let-7 family mutant phenotypes most evident in the C. elegans seam and hypodermis  after the 4th larval stage . l. llet-7, at 20°C , and ruppression  and the pression  observedhenotype . Knockdo at 15°C . The obshrpk-1 mutations on the activity of lsy-6 miRNA-dependent processes. lsy-6 regulates cell fates of two bilaterally symmetrical ASE neurons  change to [CAAGGCCGTGAACGATTCGGTCG] immediately upstream of the GFP tag sequence. Animal microinjections were performed as previously described , UY42 (hrpk-1(tm5522)), UY43 (hrpk-1(tm5522); maIs105[col-19::gfp]), OH3646 (otls114[Plim-6::gfp + rol-6(su1006)]; lsy-6(ot150)), UY46 (hrpk-1(zen17) otIs114[Plim-6::gfp + rol-6(su1006)]; lsy-6(ot150)), UY54 (hrpk-1(zen17) otIs114[Plim-6::gfp + rol-6(su1006)]), UY9 (hrpk-1(tm5522) otIs114[Plim-6::gfp + rol-6(su1006)]; lsy-6 (ot150)), UY18 (hrpk-1(tm5522) otls114[Plim-6::gfp + rol-6(su1006)]), PS3662 (syIs63[cog-1::gfp + unc-119(+)]), OH7310 (otIs193 [cog-1p::lsy-6hp + rol-6(su1006)] syIS63[cog-1::gfp + unc-119(+)]), MT14119 (mir-35-41(nDf50)), UY56 (hrpk-1(zen17)/ht2gfp; mir-35-41(nDf50)), UY55 (hrpk-1(tm5522)/ht2gfp; mir-35-41(nDf50)), VT1296  maIs105[col19::GFP]), UY67 (hrpk-1(zen17); mir-241 mir-48(nDf51) maIs105[col19::gfp]), UY68 (hrpk-1(tm5522); mir-241 mir-48(nDf51) maIs105[col19::gfp]), HML11 (maIs105[col19::gfp] lin-2 (e1309) let-7 (n2853)), UY157 (hrpk-1(zen17); maIs105[col19::gfp] lin-2 (e1309) let-7 (n2853)), VT3841 ), UY66 (hrpk-1::gfp(zen64)), MT7626 (let-7(n2853)), and UY238 ).The following strains were used in this study: N2 S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Fighrkp-1(zen15) and hrpk-1(zen17) produce similar levels of sterility (A), embryonic lethality (B), and brood size (C). An increase in embryonic lethality in hrpk-1(zen15) mutant animals (D) is most likely due the difference in the number of outcrosses between the strains, with hrpk-1(zen17) being outcrossed seven times, while hrpk-1(zen15) was outcrossed only twice. ***p≤0.001.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fighrpk-1(zen17) and hrpk-1(tm5522) alleles reveal that hrpk-1 activity has a maternal component for some developmental processes such as fertility (A), brood size (B), and embryonic viability (C), but not for vulval integrity in day 3 or older adults (D). hrpk-1(tm5522) appears to be weakly semi-dominant as evidenced by the presence of defects observed in hrpk-1(tm5522)/+ and hrpk-1(tm5522)/hrpk-1(zen17) animals (A-D). Genotype of the score animals is shown. m- indicates that scored animals came from homozygous mutant mothers, m+ indicates that scored animals were progeny of wild type mothers. hrpk-1(tm5522)m/hrpk-1(zen17)p animals came from a cross between hrpk-1(tm5522) mothers and hrpk-1(zen17) fathers. hrpk-1(tm5522)p/hrpk-1(zen17)m animals came from hrpk-1(zen17) mothers and hrpk-1(tm5522) fathers.Genetic analyses of (TIF)Click here for additional data file.S4 FigA) or embryonic viability (B).C-terminal HRPK-1 GFP tag does not affect animal fertility ((TIF)Click here for additional data file.


Staphylococcus aureus (MRSA) strains cause life-threatening diseases. We present a draft genome sequence of PVL-positive MRSA sequence type 154 (ST154) strain NRL 08/001, isolated from a fatal case of necrotizing pneumonia. The genome consists of 2.9 Mb over 39 contigs and harbors novel composite island staphylococcal cassette chromosome mec element (SCCmec)-mercury composite type 2B&5.Panton-Valentine leucocidin (PVL)-positive methicillin-resistant  Staphylococcus aureus (MRSA) strains cause life-threatening diseases. We present a draft genome sequence of PVL-positive MRSA sequence type 154 (ST154) strain NRL 08/001, isolated from a fatal case of necrotizing pneumonia. The genome consists of 2.9 Mb over 39 contigs and harbors novel composite island staphylococcal cassette chromosome mec element (SCCmec)-mercury composite type 2B&5.Panton-Valentine leucocidin (PVL)-positive methicillin-resistant  Staphylococcus aureus (MRSA) clones will help advance our understanding of the pathogenic potential, evolution, and spread of such superbugs -positive strains are often linked with hemorrhagic necrotizing pneumonia . Sequencuperbugs .S. aureus strain NRL 08/001 (=CNCTC 7452), belonging to sequence type 154 (ST154) and spa type t667  (int prophage carrying the PVL locus (lukS-PV and lukF-PV), a 42.2-kb-long Sa3int prophage harboring immune evasion cluster type B (int prophage disrupted by the insertion of a 13.1-kb transposon upstream of the terS gene. The SCCmecFinder v1.2 (mec element (SCCmec)-mercury type 2B&5 composed of SCCmec type IV, an SCC with ccrC recombinase, and a mercury resistance operon usually found in SCCmercury adjacent to SCCmec III (S. aureus pathogenicity island (SaPI) carrying genes for enterotoxins C and L inserted into the tmRNA binding protein gene (smpB), (ii) a 7.0-kb-long incomplete SaPI with genes encoding a ferrichrome-binding periplasmic protein (fhuD) and a potassium uptake protein (trk), and (iii) an 18.0-kb-long conjugal transposon, Tn916, with the tetM resistance gene. Plasmid DNA was found on 5 contigs.Bacteria were cultured for 18 h at 37°C in 2× YT medium , and genomic DNA was extracted using a High Pure PCR template preparation kit (Roche) with 5 mg/ml lysostaphin added to the suspension buffer. A 400-bp sequencing library was constructed using an Ion Plus fragment library kit (Thermo Fisher Scientific) and sequenced on the Ion Torrent PGM (Thermo Fisher Scientific) using an Ion 318 Chip v2. This generated 5,084,145 single-end reads, for a total of 1.7 Gb, with a mean read length of 335 bp. Default parameters were used for all software, unless otherwise stated. FastQC v0.10.1 was used for read quality assessment. The raw reads were error corrected and assembled using SPAdes v3.13.0  L50 and NVHNE00000000 (SRA accession no. SRR9600155).This whole-genome shotgun project has been deposited in GenBank under accession no.


Inhibition of PRMT5, the major Type II arginine methyltransferase, suppresses pathogenic T cell responses and EAE. PRMT5 is transiently induced in proliferating memory inflammatory Th1 cells and during EAE. However, the mechanisms driving PRMT5 protein induction and repression as T cells expand and return to resting is currently unknown. Here, we used naive mouse and memory mouse and human Th1/Th2 cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Initial activation of naive mouse T cells resulted in NF-κB-dependent transient Prmt5 transcription and NF-κB, mTOR and MYC-dependent PRMT5 protein induction. In murine memory Th cells, transcription and miRNA loss supported PRMT5 induction to a lesser extent than in naive T cells. In contrast, NF-κB/MYC/mTOR-dependent non-transcriptional PRMT5 induction played a major role. These results highlight the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS.Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) mediated by CD4 Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), thought to be driven by myelin-reactive inflammatory T cells. T cell responses against myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) antigens are thought to underlie MS. In support of this notion, immunization against these antigens induces the MS-like disease experimental autoimmune encephalomyelitis (EAE) in mice and other rodents –3. MS anWe recently reported that protein arginine methyltransferase 5 (PRMT5) plays a crucial role in inflammatory T cell expansion and EAE disease . PRMT5 iPRMT5-targeting miR-96 and miR-92b in Epstein Barr Virus-transformed cell lines was driven by nuclear factor (NF)-κB signaling and resulted in PRMT5 protein induction , phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) pathways. The Erk pathway drives transcription and translocation of transcription factor Fos into the nucleus, which together with Jun, forms the functional Activator Protein 1 (AP-1) complex. Transcription factors NF-κB and AP-1 converge to rapidly upregulate IL-2 expression, a growth, and survival cytokine that drives T cell expansion , 25. PI3ys in MS –44. In ays in MS . Given tys in MS , 22 as wPrmt5 transcription and PRMT5 protein induction, the latter mediated by MYC induction and mTOR-induced miR-322 loss. In contrast, in memory Th cells, the NF-κB/MYC/mTOR axis was dispensable for Prmt5 transcription and loss of Prmt5-targeting miRNAs. Rather, NF-κB/MYC/mTOR pathways contributed to PRMT5 protein induction via non-transcriptional/non-miRNA mechanisms. Overall, these data are consistent with a model in which initial murine naive T cell activation induces PRMT5 via both transcriptional and non-transcriptional mechanisms downstream of NF-κB, mTOR and/or MYC. During recall activation, memory T cells would be poised to rapidly induce PRMT5 expression, mainly through NF-κB/MYC/mTOR-dependent non-transcriptional mechanisms, possibly allowing the more robust proliferative response characteristic of memory T cell responses. Overall, this study provides insight into the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic Th cell responses and may guide targeted therapeutic strategies for MS.In this study, we explore the signaling pathways and mechanisms driving PRMT5 expression after T cell activation. Using murine naive and memory as well as human memory Th cells as models of initial and recall T cell activation, we show that PRMT5 protein expression is regulated via a combination of transcriptional and non-transcriptional mechanisms. NF-κB, mTOR and MYC pathways promoted PRMT5 protein induction in murine naive and memory T cells. However, some differences in the mechanisms of PRMT5 regulation were observed between naive and memory T cells. In naive T cells, NF-κB induced both Ac1−11-specific TCR-transgenic (Tg) mice [described previously (B10.PL (Jackson Laboratory) and myelin basic protein (MBP)eviously ] were brin vitro studies.Stock Bay11-7082 or Bay11-7085 (SelleckChem), 10058-F4 (Sigma or SelleckChem), LY294002 (SelleckChem), SCH772984 (SelleckChem), and Rapamycin (SelleckChem) were solubilized in DMSO vehicle and diluted ≥1:1,000 for Ac1−11 and irradiated splenocytes in the presence of recombinant human (rh) IL-2 (Miltenyi) every 7–10 days. T cells collected 7–10 days after activation with MBPAc1−11 and irradiated splenocytes provided the resting Th cell condition. To avoid the presence of non-T cells in in vitro experiments, resting Th1 or Th2 cell lines were activated with anti-CD3/CD28 for the indicated lengths of time. Mouse naive CD4+ T cells were isolated from spleens and lymph nodes using the mouse naive CD4+ T cell isolation kit (Miltenyi or STEMCELL Technologies) and activated using 5 μg/ml coated anti-CD3 and 2 μg/ml soluble CD28. Human Th1 and Th2 cells were generated by isolating CD4+ T cells with a CD4+ T Cell Isolation Kit (STEMCELL Technologies) from human whole blood leukocytes from normal donors and activating on anti-CD3/CD28 Dynabeads (Thermo Fisher) under Th1 (rhIL-12 + anti-hIL-4) or Th2 (rhIL-4 + anti-hIL-12 + anti-hIFNγ) conditions for 1 week and reactivated for further experiments, as previously described , according to the manufacturer's instructions, and stored at −80°C until analysis. Optical density (OD) 230, 260, and 280 were obtained with a NanoDrop 2000 to evaluate RNA concentration and quality.Prmt5 (Mm00550472_m1), mouse Hprt (Mm0044968_m1), mouse Myc (Mm00487804_m1) or human PRMT5 (Hs01047356_m1) and human 18S (Hs99999901_s1) primer sets (Life Technologies), as previously described  or random primers and Superscript III (Applied Biosystems) according to manufacturer's instructions; TaqMan quantitative real-time PCR was performed using mouse 5 Mm005502_m1, mou5 Mm005502_m1, mouPrimary naive or memory T cells were transfected with Mirus TransIT-TKO (MIR2150) or TransIT-X2 (MIR6003) transfection reagent according to manufacturer's instructions. Briefly, cells were activated for 4–8 h and subsequently incubated with 25–50 nM of negative control miRNA mimic (4464061 or AM17111), or a combinations of miR-15b (MC10904), miR-140-3p (MC12503), and/or miR-322/424 (MC11080) mimics. Average transfection efficiencies, measured by transfection with Cy3 conjugated negative control miRNA (AM17120), were 30% for naive Th cells, 15–20% for memory Th1 cells and 50% for memory Th2 cells.Prmt5 or human PRMT5 3′-UTR. In some cases, mouse Prmt5 3′UTRs with mutated miRNA-binding sequences were transfected. Mutated 3′UTRs were ordered from IDT. Mouse Prmt5 3′UTR was mutated as follows: The miR-140-3p binding site CUGUGGA was mutated to GCCGCCG, the miR-15/16/195/322 binding site UGCUGCU was mutated to CCGGCGC, and the miR-96 sites UGUAGAACAUCUGCUGGUUCAGU and UGCUCAGCCGCCAGA were deleted. Human PRMT5 3'UTR was mutated at the putative miR-140-3p binding site CCCUGGA to GCCGCCG. The wild-type or mutant plasmids were co-transfected with miR-15a (MC10235), miR-15b (MC10904), miR-16 (MC10339), miR-96 (MC10422), miR-140-3p (MC12503), miR-195 (MC10827), murine miR-322/424 (MC11080), or human miR-424 (MC10306), or control nonsense (NS) pre-miR (4464061 or AM17111) (Thermofisher). Cells media was changed to complete media  + Penicillin/Streptomycin) after 24 h. Cells were collected and lysed in 1X Cell Lysis Buffer (Promega) 4 days post transfection. Luminol was detected using a GloMax 96 Microplate Luminometer (Promega). Firefly luciferase signal was normalized to Renilla luciferase as a measure of transfection efficiency.Cos-7 cells were transfected using Lipofectamine (Life Technologies) in serum-free conditions with pmiRGlo Dual Glow Luciferase plasmid (Promega) containing the wild-type mouse Cells were collected at different time points and cell pellets were frozen at −80°C. Cell pellets were lysed in RIPA buffer  and protein concentration was quantified by BCA assay. Protein (5–10 μg) was run on 14% SDS-PAGE gels and transferred onto PVDF membrane. Blots were blocked with Odyssey Blocking Buffer (LICOR) and probed with antibodies against PRMT5 (Abcam ab31751), SYM10 pan-symmetric dimethylarginine (Millipore 07–412), or MYC . β-actin (Sigma) was used as a housekeeping control. Secondary antibodies used were goat anti-rabbit 800CW and goat anti-mouse 680RD (LICOR). Blots were imaged on an Odyssey CLx machine (LICOR) and quantifications were performed using ImageStudio.We previously reported that PRMT5-selective inhibitors suppressed not only memory Th cell proliferation, but also the proliferation of newly activated naive Th cells, albeit higher concentrations were required . This suTo explore which TcR-induced pathways contribute to PRMT5 induction, we targeted major pathways activated via the TcR, namely NF-κB, PI3K, mTOR, and Erk. Out of these, NF-κB has been shown to promote PRMT5 in cancer cells . We founMyc transcript  reduction in PRMT5 protein expression in activated memory Th cells  4-mediated translation initiation , 57, 58 ependent  and IRESependent . While tPrmt5/PRMT5-targeting miRNAs in mouse and human T cells. In the early phases after naive T cell activation, only miR-322/424 decreased significantly, suggesting that most of PRMT5 induction occurs via the transcriptional and non-transcriptional mechanisms described in the paragraph above. However, miR-322/424, miR-15b, and miR-140-3p substantially increase as naive T cells down-regulate PRMT5 and return to the resting non-proliferative state of resting memory cells, suggesting these miRNAs contribute to PRMT5 suppression. Once these resting memory T cells were reactivated, Prmt5-targeting miRNAs decreased, which may contribute to PRMT5 protein induction and fast proliferative responses in recall memory T cell responses. Overexpression of Prmt5-targeting miRNAs in our system resulted in modest decreases in PRMT5 protein. miRNAs suppressing PRMT5 protein expression would be expected to suppress T cell proliferation as well. One limitation is that, since efficient transfection of primary Th cells is difficult to achieve, a majority of untransfected cells continue to proliferate, overwhelming the culture and making it difficult to observe an effect. Thus, it is conceivable that miRNAs may have a larger effect on PRMT5 protein expression than our data may initially suggest.Post-transcriptional miRNA-mediated regulation of PRMT5 expression has long been known to operate in transformed cells , 21 and MYC. Although the implications of this SNP on MYC activity and PRMT5 expression remain to be fully elucidated, we show here that MYC activity is required for full PRMT5 induction in T cells. Finally, miRNAs have also been shown to be dysregulated in MS patients  and the Policy on Humane Care and Use of Laboratory Animals (PHS). Animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Studies using human T cells were all completely de-identified and therefore, exempt from Institutional Review Board (IRB) approval.LW, JN, SA, and MG-d-A formulated research hypothesis and designed experiments. LW, JN, SA, SS, and GN performed experiments and interpreted data. LW drafted the manuscript. MG-d-A revised manuscript. All authors extensively reviewed the manuscript.MG-d-A has a PRMT5 inhibitor patent pending and is a PRMT5 inhibitor inventor on a licensing deal with Prelude Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila wing epithelium, Wingless  is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. Here, we performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo ‘temporal-rescue’ assay, our results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, our results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells.Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the  Drosophila wing as a model system to study Wnt/Wg secretion, the authors performed a large-scale RNAi screen and identified proteins of the exocyst complex to be required for Wnt signaling. The study shows that exocyst complex preferentially regulates apical secretion of Wg proteins. Taken together, this study identifies routes and regulators for secretion of signaling-active Wnt proteins from polarized epithelial cells.Regulation of Wnt signaling and the production of Wnt ligands is crucial for proper development and homeostasis, as dysregulation leads to developmental defects and diseases such as cancer. This study addresses the question of how functional Wnt ligands are secreted by epithelial cells. By using the polarized epithelium of the developing  Drosophila,5, Wnt ligands and their downstream signaling cascades have been subsequently studied in different organisms and many evolutionarily conserved genes required for signaling upstream and downstream of the receptors have been identified [in vivo and in cultured Drosophila cells, have led to the discovery of components of the Wnt secretion pathway, including the ER membrane protein Porcupine [Wnts are secreted lipid-modified proteins conserved in all animals and play a pivotal role in development and many human diseases such as cancer –3. Initientified ,7. Subseorcupine , the Wntorcupine –11, p24 orcupine ,13 and torcupine –19. Thesorcupine ,21. In cDrosophila wing imaginal discs, which give rise to the adult wing and thorax, are made up of tightly packed columnar epithelial cells (disc proper cells) and a squamous epithelial sheet towards the apical side of disc proper cells called the peripodial membrane [membrane . In the membrane –25, whilmembrane ,27, depemembrane . In contmembrane , which rin vivo RNA interference (RNAi) screen to identify regulators of Wg secretion from polarized epithelial cells. The screen identified components of the exocyst complex to be required for polarized Wg secretion. The exocyst complex has been implicated in different cellular trafficking processes, including polarized secretion, ciliogenesis and cell migration [So far, the route of Wg secretion from polarized epithelia has mostly been traced by either following a fluorescently tagged exogenous Wg protein ,30 or byigration –33. Moleigration . We findin vivo. We assembled a library of 394 RNAi lines targeting genes with a putative role in protein transport  and achaete [Distal-less (Dll) [sec6 or evi RNAi constructs under the control of apterous-Gal4. Apterous (ap) is expressed in the dorsal compartment of the imaginal disc and its expression overlaps with only one out of two rows of Wg producing cells. Previous studies have demonstrated that Evi is not required in the receiving cells for signal transduction [ap-Gal4>evi RNAi led to an accumulation of Wg in the dorsal row of producing cells whereas Sens–due to cell non-autonomous rescue by Wg protein secreted from the ventral row of Wg producing cells–remained expressed  –43. To asduction ,10. Consxpressed . Similarxpressed . This deng cells , arrows. of Sens , arrow a of Sens . Taken tin vivo ‘temporal-rescue’ experiment whereby we block Wg secretion in evi2 homozygous null mutants and then relieve this block by expressing an Evi-mCherry rescue construct under the control of a temperature sensitive Gal4/Gal80ts expression system  until the third instar stage and then shifted to 29°C (the permissive temperature for expression) for 7, 16 and 25 hours (h)  . Expressours (h)  and initours (h) . Concurrours (h)  with peaours (h) , which wours (h) . Analysiours (h)  as compaours (h) . Taken tsens and Dll in a ‘temporal-rescue’ experiment. We observed that at 0h of Evi-mCherry expression, as expected Wg accumulated in the producing cells  and high-threshold (sens) target genes in the receiving cells, supporting the importance of this apical Wg pool. However, these experiments do not rule out that a small amount of Wg observed at the basolateral side can also activate low-level signaling.Our experiments provide evidence for the secretion of endogenous untagged Wg from the apical surface. The efficient block of Wg secretion in homozygous pical Wg  and withpical Wg  or by itpical Wg . We furtin vivo RNAi screen, we first screened for an overall wing margin phenotype. While this phenotype could arise from defects in processes not directly related to Wg secretion, it allowed us to shortlist genes important for wing margin formation. From further analysis, we showed that the exocyst complex is required for apical Wg secretion. The exocyst is believed to act in tethering of secretory vesicles to the plasma membrane and is localized at the sites of exocytosis in several cellular contexts [In an contexts . Consistcontexts . Interescontexts ,12,13, wsens. These results also support the previous observation that the apical localization of wg transcript is required for proper Wg signaling activity. It was shown that mis-localizing wg mRNA to the basolateral side slightly enhanced basolateral distribution, however it led to reduced Wg signaling activity [We found that depletion of exocyst complex proteins led to the loss of extracellular Wg at the apical side of the epithelium, with a concomitant increase in basolateral secretion. This basolateral increase in Wg is not observed in the absence of Evi, where Wg remains trapped in the early secretory pathway ,21, theractivity .Moveover, in support to our model, a recent study showed that the exocyst complex regulates apical secretion of Wnt1 in polarized Madin-Darby canine kidney (MDCK) epithelial cells , indicatAn important question is why apical and basolateral Wg show differential signaling strengths. Several models could explain this differential signaling activities in the polarized wing epithelium: (i) in wing discs, the repertoire of Wnt receptors and co-receptors show polarized localization. A previous study suggested that the basolateral Wg may signal via the Wnt receptor Frizzled2 (Fz2), which is found basolaterally at steady-state . HoweverTaken together, our study provides a mechanism for the secretion of endogenous Wg from polarized epithelial cells and identifies exocyst members required for apical secretion. It will be of further interest to analyze the role of the exocyst in secretion of Wnt proteins in other developmental and niche/stem cell contexts.Drosophila stocks were used: wg-GAL4 , ap-GAL4 2nd chr., [en-GAL4, UAS-GFP 2nd chr. [CyO-wg-LacZ [UAS-evi-PB-mCherry, UAS-GFP-wg [evi2, FRTG13 sec6ex15/CyO-act-GFP, y,w, hs-flp; FRTG13 Ubi-GFP M(2)58F/CyO (a gift from X. Lin), y,w, hs-flp; FRTG13 ubi-GFP.nls/CyO (a gift from Y. Bellaiche), dpp-Gal4/TM6b (Bloomington stock center stock # 7007), ubi-GFP FRT2A (A. Gould). UAS-sec6-RNAi (KK-ID 105836 and GD-ID 22077), UAS-evi-RNAi (KK-ID 103812), UAS-sec15-RNAi (KK-ID 105126), UAS-sec3-RNAi (KK-ID 108085), UAS-Rel-RNAi (KK-ID 108469) and UAS-wg-RNAi (GD-ID 13352) lines were obtained from the Vienna Drosophila RNAi Center [The following nd chr., , en-GAL42nd chr. , CyO-wg--wg-LacZ , UAS-eviS-GFP-wg , evi2[1i Center . All croin vivo RNAi screen was performed using the wg-GAL4 driver to express RNAi transgenes . Crosses were reared at 25°C.Genes involved in intracellular transport were selected as previously described in . The in in vivo ‘temporal-rescue’ experiments of evi2 mutants shown in Figs en-Gal4, UAS-GFP/UAS-evi-mCherry; evi2, tub-Gal80ts/evi2 at 18°C to prevent Evi expression  until third instar stage and then shifted to 29°C , to induce GFP and Evi-mCherry expression. Larvae were shifted to 29°C for 7, 8, 16 or 25 hours before dissection.The y,w, hs-flp; FRTG13 ubi-nlsGFP/FRTG13 sec6ex15 or y,w, hs-flp; FRTG13 Ubi-GFP M(2)58F/ FRTG13 sec6ex15 genotype, for one hour at 37°C during first- and second-instar larval stages. Wing imaginal discs from wandering third-instar larvae were dissected for antibody staining.Sec6 loss of function clones were generated by heat shocking larvae of either The following genotypes were used in this study:wg-Gal4/UAS-Rel-RNAi (KK)wg-Gal4/UAS-sec6-RNAi (KK)wg-Gal4/UAS-evi-RNAi (KK)en-Gal4, UAS-GFP/UAS-sec6-RNAi (KK)en-Gal4, UAS-GFP/UAS-evi-RNAi (KK)y w hs-FLP/+; FRTG13 sec6ex15/FRTG13 Ubi-nlsGFPw1118en-Gal4, UAS-GFP/UAS-evi-mCherry; evi2, tub-Gal80ts/evi2en-Gal4, UAS-GFP/UAS-sec3-RNAi (KK)en-Gal4, UAS-GFP/UAS-sec15-RNAi (KK)UAS-sec6-RNAi (KK)/ CyO-wg-LacZ; dpp-Gal4/+wg-Gal4, UAS-GFP-wg/UAS-Rel-RNAi (KK); tub-Gal80ts/+wg-Gal4, UAS-GFP-wg/UAS-sec6-RNAi (KK); tub-Gal80ts/+wg-Gal4, UAS-GFP-wg/UAS-evi-RNAi (KK); tub-Gal80ts/+en-Gal4/UAS-sec6-RNAi (GD)ap-Gal4/+; UAS-GFP/+UAS-evi-RNAi (KK); UAS-GFP/+UAS-sec6-RNAi (KK); UAS-GFP/+y w hs-FLP/+; FRTG13 sec6ex15/ FRTG13 Ubi-GFP M(2)58FThe following antibodies were used: Mouse Anti-Wg  1:5 for extracellular and 1:50 for total staining, Rabbit-anti-Dll 1:200 (a gift from S. Carroll), Guinea Pig anti-Sens 1:300 , Guinea Extracellular Wg staining was performed as described previously . Wing diwg-GAL4, UAS-GFP-wg; tub-Gal80ts flies were crossed to flies containing either UAS-Rel-RNAi or UAS-evi-RNAi or UAS-sec6-RNAi. Crosses were reared at 18°C until early larval stages and were then shifted for 72 hours at 29°C for expression. Wing imaginal discs were imaged using a Leica SP5 confocal and images were generated using ImageJ software [software . Secretesoftware .S1 Fig(A–A”) Depletion of another exocyst component Sec3 in the posterior compartment using en-Gal4, UAS-GFP shows accumulation of Wg inside the producing cells. (A”) The graph shows fold change in the mean intensity between control Wg levels  and Wg levels in sec3 RNAi (GFP positive), which are higher than the control (N = 7). (B–B”) Depletion of Sec15 in the posterior compartment (GFP positive) of the discs shows accumulation of Wg inside the producing cells. (C–C”)sec6 RNAi was expressed using dpp-Gal4 (double sided arrows show dpp expression domain) in wg-LacZ background. No change is observed in the levels of LacZ expression (C’) while Wg accumulation can be observed (C”). (D–E) GFP-Wg was expressed along with control  Relish (Rel) RNAi and evi RNAi or sec6 RNAi. Quantification shows significant reduction in the secreted GFP-Wg puncta in sec6 RNAi compared to the control Rel RNAi, while evi RNAi showed complete loss of GFP-Wg secretion. N = 4 for D and E. error bars s.d.. P = 0.003 generated by Student’s t-test. Scale bar 20 μm.(TIF)Click here for additional data file.S2 Fig(A–B)en-Gal4, UAS-GFP/UAS-sec6 RNAi (GD) was used to deplete Sec6 in the posterior compartment of the discs (GFP positive). Extracellular Wg staining performed on these discs show reduced apical level (A–A’) while the basolateral levels were increased (B–B’). Scale bar 20 μm.(TIF)Click here for additional data file.S3 Fig(A) Extracellular Wg staining on disc with Sec3 depletion in the posterior compartment show reduced apical Wg, (A”) graph shows normalized mean intensity showing reduced levels of extracellular apical Wg in the RNAi domain (N = 3). (B–B’) Enlarged region in A (marked with white box) shows clonal expression of GFP. The GFP negative cells show normal levels of extracellular Wg apically . (C–C”) The basolateral levels of the extracellular Wg were increased in the sec3 RNAi expression domain; (C”) graph shows normalized mean intensity quantification as above (N = 3). (D–D”) Extracellular Wg staining on discs with Sec15 depletion show reduced apical Wg levels. (E–E”) While basolateral Wg levels were mildly increased in the posterior sec15 RNAi compartment (D”–E”) Graph shows normalized mean intensity quantification as above (N = 5). Scale bar 20 μm, error bars: s.d.(TIF)Click here for additional data file.S4 Fig(A–A”) Control discs showing expression domain of ap-Gal4/UAS-GFP with Sens and Wg staining. (B–B”)evi RNAi expressed with ap-GAL4 shows Wg accumulation in one  row of producing cells (B and B”) and Sens expression is still observed in the dorsal side of the DV boundary (B’). (C–C”) Similarly Sec6 depletion shows Wg accumulation in dorsal row of cells while Sens expression is observed. (D–D’) Sens staining on disc with sec6ex15 clones (in heterozygous Minute background) in the Wg producing cells (yellow dotted line) show reduced Sens expression around the clones (arrows). (E–E’)sec6ex15 clones in the Wg receiving cells (yellow dotted line) shows expression of Sens (arrow). (F–F’)sec6ex15 clones show normal expression of Dll. Scale bar 20 μm. (N = 3 minimum).(TIF)Click here for additional data file.S5 Figen-Gal4, UAS-GFP/UAS-evi-mCherry; tub-Gal80ts, evi2/ evi2 discs were kept at 18°C until the third instar stage and then shifted to 29°C for 7 hours (h), 16h and 25h. Extracellular Wg staining was performed on these discs. Panels in (A) show 7h expression of GFP (green) and Evi-mCherry (red), where low-level expression of both GFP and Evi-mCherry can be seen . Extracellular Wg staining shows higher levels of Wg at the apical side as compared to the basolateral . (B) Similarly, panels in B show another example of 16h of expression  Similarly, 25h after expression. Graphs show mean intensity of the extracellular Wg staining across the dotted white line. (N = 3 minimum for each panel), AP = Anterior-Posterior boundary, a.u. = arbitrary unit. Scale bar 20 μm.(TIF)Click here for additional data file.S6 Fig(A–A””) 0 hour of Evi-mCherry expression, where almost no expression of Evi-mCherry (red) and GFP (green) can be observed (A–A’) and Wg accumulation in the posterior compartment of the evi2/evi2 discs can still be observed (A”). Sens staining on these discs show no rescue of Sens expression near DV boundary (A”’–A””). (B–B””) 7 hours after temperature shift where very weak expression of Evi-mCherry and GFP is observed (B–B’), while total Wg still remained unchanged (B”) and Sens expression at the DV boundary remained undetectable (B”’–B””). (C–C”) After 16 hours a strong expression of Evi-mCherry and GFP can be observed, moreover Wg accumulation was rescued in the expression domain. (C”’–C””), Weak expression of Sens at the DV boundary (posterior compartment) appears at 16 hours rescue. (D–D””) 25 hours of Evi expression shows similar rescue of both Wg accumulation and Sens expression. N≥3 wing discs for each panel; Scale bar 20 μm.(TIF)Click here for additional data file.S1 Tablen vivo RNAi screen for Wg secretion. All RNAi lines (KK or GD) were crossed with wg-GAL4 and crosses were kept at 25°C. Adult wings were scored for notch phenotypes. Six out of eight exocyst complex genes showed notch phenotype (marked in red) and they were identified as a strong candidate for Wg secretion defect. One component (Sec10) was not contained in our library and Exo70 did not give a phenotype.The above shown genes were selected for i(PDF)Click here for additional data file.


Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of   •Intestinal carbohydrate metabolism is male-biased and region-specific•Testes masculinize gut sugar handling by promoting enterocyte JAK-STAT signaling•The male intestine secretes citrate to the adjacent testes•Gut-derived citrate promotes food intake and sperm maturation Inter-organ communication couples diet with gamete production. The male gonad promotes sex differences in carbohydrate metabolism within an adjacent intestinal portion via JAK-STAT signalling. In response to this gonadal signal, gut-derived citrate controls food intake and sperm maturation. Males and females differ in their physiology and disease susceptibility  yet the Drosophila melanogaster activates a splicing cascade that results in expression of the RNA-binding protein TraF only in females   see  and publ-A7) see A–1I. The-A7) see D.Figure Mal-A7, and Pgi; quantification is shown in Mal-A7, respectively). Sexual dimorphism in the R4 region was not restricted to carbohydrate metabolism genes; it also included oxidative stress response genes such as Glutathione S transferase D1 (GstD1)  E and gen (GstD1) F.Thus, the proteins handling sugars in the adult gut are male-biased, and this intestinal sexual dimorphism is spatially confined to the posterior R4 midgut region.tra (F and its binding partner Transformer 2 (Tra2) (tra and tra2 mutation (masculinization) or traF mis-expression (feminization) on the subset of gut-specific, male-biased sugar genes  that allows whole-body or cell-type-specific tra deletion and a traF K-IN knockin allele that constitutively feminizes males. This allele fully rescues tra-null mutant females (unlike UAS-traF), including their fertility  flies  flies to amounts comparable to those detected in female guts   by assesar genes A–2C. We lity see A–S2D. Boale guts B.Figure F would control sex differences in intestinal sugar genes intrinsically from the intestinal epithelium itself, like the sex differences in intestinal stem cell proliferation  lines and the tra allele that allows its cell-type-specific deletion  sexual identity of adult intestinal progenitors controls their female-biased proliferation , we examined the spatial restriction of the intestinal sexual dimorphism in sugar gene expression in more detail—in particular, its three-dimensional arrangement inside the male body cavity. Immunohistochemical analysis of the internal organs in their intact arrangement see  revealedsans fille (snf) mutant female flies, or in female flies with germline-specific Sex lethal (Sxl) or snf KDs, which resulted in de-repression of testis genes in the “female” gonad     B and 3C.shutoff (esgSHOF) mutant male flies lacking a functional testis (zero population growth (zpg) mutants, which lack the male germline but have an intact somatic hub  , pathwayStat92E-GFP)   displaye92E-GFP) C and 4D,92E-GFP) E. A cand92E-GFP) . Upd1 is92E-GFP) . Downregale guts B, althousnf or nanos (nos) > Sxl-RNAi females in which the transformed ovaries ectopically activate JAK-STAT ligands and pathway components ; and (2) ectopically activated the JAK-STAT pathway in female ECs by expressing a constitutively active Hopscotch (Hop) (UAS-hopTum) or the JAK-STAT ligand Unpaired 3 (Upd3) from midgut expression 1 (mex1)-Gal4. In both cases, intestinal sugar gene expression was upregulated in female guts  in males, or ectopically activated JAK-STAT signaling in females. Clones with reduced JAK-STAT signaling in males downregulated the Amy-p reporter in R4  or exacerbating (upd3 overexpression) JAK-STAT signaling in ECs of the midgut R4 region. We also reduced the male bias in JAK-STAT signaling independently from the male gonad in two ways: by depleting the testis from hub cells in esgshof males (upd1 from the hub by using fascilin 3 (fas3)-Gal4 (expressed in the hub cells of the testis)  . Using f testis) , we obse testis) I. Thus, UAS-FLII12Pglu-700μδ6) (UAS-based version of the laconic sensor)   and a la sensor)  (see STA sensor) . The glu sensor) A, consisTo monitor lactate levels, we used our validated lactate reporter D to showTo test this idea, we used food intake as a behavioral readout for a genetic screen in which we knocked out male-biased intestinal sugar genes, reasoning that KD of any enzymes mediating conversions “upstream” of this metabolite or those involved in its transport out of the EC would reduce food intake, whereas KD of “downstream” enzymes would have no effect . R2- and R4-specific KD of genes for enzymes involved in sugar digestion, absorption, and glycolysis  for mitochondrial oxidation see , all faiDrosophila)  (kdn or PCB (A third way in which pyruvate is utilized involves the anaplerotic pyruvate carboxylase (PCB)-mediated pathway leading to citrate production through the pyruvate/citrate cycle , and invsophila) . Genetic1 (Mpc1)  KD, expen or PCB  KD, redun or PCB F and 6G.n or PCB , which wn or PCB C and S6Fn or PCB , which wn or PCB C and S6FATP citrate lyase (ATCPL) (I’m not dead yet (Indy) transporter   , which c (ATCPL) F and 6G.nsporter , known tnsporter , reducednsporter F and 6G.lt males J. To cons (CIT8)  (see Mets (CIT8)  and valis (CIT8)   . Using ts (CIT8) C. Monitoe R4 ECs C, confirIndy knockdown) did not affect male stem cell proliferation , which reduced food intake without reducing stem cell proliferation to basal levels  had no effect on their feeding behavior   (GFP) labeling of primary spermatocyte nuclei  and capillary electrophoresis mass spectrometry (CE-MS) see , and obsIndy KD) . CE-MS a(Indy KD . We hypo3 [pH3])   B. Howeve3 [pH3]) , pH3 num3 [pH3]) B. This we nuclei , which r-driven Indy KD) impaired spermatogenesis without affecting food intake , comparing adult testes from control male flies to those from male flies in which the Indy citrate efflux transporter had been specifically downregulated in intestinal ECs of the R2 and R4 regions. We observed a trend toward reduced citrate levels in testes samples after intestinal knockdown, consistent with reduced exogenous supply of citrate to the testis . ImpaireMal-A1RNAi    (and dat testes) I but not testes) L or earl testes) M (for te testes) B and S7NDrosophila and whether the evolution of a placenta (an organ purpose-built for reproduction) replaced or reinforced such intestinal contributions in female mammals.Regional differences in gene expression are observed along animal gastrointestinal tracts, suggestive of functional specializations . We now Drosophila Upd belong to the type I family of cytokines, like mammalian interleukins and leptin. In both humans and rodents, leptin expression is sexually dimorphic .CG7309 and Indy-2 genes code for putative citrate transporters and have testis-specific expression  should be directed to and will be fulfilled by the Lead Contact, Irene Miguel-Aliaga .Further information and requests for resources and reagents . All experimental flies were kept in incubators at 25°C, 65% humidity and on a 12 hr light/dark cycle. Flies were transferred to fresh vials every 3 days, and fly density was kept to a maximum of 15 flies per vial. 5-day old virgin flies were used unless otherwise indicated.For metabolomics and testes immunostainings, males were aged for 10 days before dissection. For clonal analyses (flip-out clones), 3-day-old adults (raised and aged at 25°C) were heat-shocked for 12 min at 37°C to induce clones, and were then kept at 25°C for 5 days until dissection. Flies were transferred to fresh vials every 3 days.GFP (VDRC: 318296), Mal-A3GFP (this study), TrehGFP (BDSC: 59825), Hex-AGFP (VDRC: 318587), PgiGFP (this study), Amy-pGFP (this study), LdhGFP , GstD1-GFP , ComrGFP (VDRC: 318559), BouleGFP (BDSC: 64431), bamGFP (VDRC: 318001).Mal-A1R2R4-Gal4  followed by a sequence encoding a Gal4 driver, , mex1-Gal4 , nos-Gal4 (BDSC: 4937), Act5C-Gal4 (BDSC: 4414), tj-Gal4 (DGGR: 104055), Act5C-FRT-y-FRT-GAL4 (BDSC: 4410), Fas3-Gal4 (DGGR: 103948), da-Gal4 (BDSC: 55851), prosV1-Gal4 , vm-Gal4 (BDSC: 48547), elav-Gal4 (BDSC: 458), repo-Gal4 (BDSC: 7415), Lpp-Gal4 , Akh-Gal4 (BDSC: 25683), Aug21-Gal4 (BDSC: 30137), eya-Gal4 , R2R5-Gal4 , esg-Gaex1-Gal4 , Mal-A7-sV1-Gal4 , Myo1A-GLpp-Gal4 , Hml-GalUAS-StingerGFP (BDSC: 65402), UAS-mCD8GFP , UAS-FLII12Pglu-700μδ6 , UAS-Ldh (FlyORF: F002924), UAS-flp (BDSC: 4539), UAS-traF (BDSC: 4590), UAS-upd3-GFP , UAS-domeΔCYT (UAS-dcr2 (VDRC: 60010), UAS-InRDN (BDSC: 8252), UAS-gRNAS Amy-p/Amy-d/Mal-A1 , UAS-Cas9 (BDSC: 54594), UAS-LdhRNAi (BDSC: 33640), UAS-traRNAi (BDSC: 28512), UAS-snfRNAi (BDSC: 55914), UAS-SxlRNAi (BDSC: 38195), UAS-GFP (BDSC: 35786), attP2 control line (BDSC: 36303), attP40 control line (36304), GD control line (VDRC: 60000), KK control line (VDRC: 60100), UAS-domeRNAi (BDSC: 34618), UAS-hopRNAi (VDRC: GD 40037), UAS-Stat92ERNAi (BDSC: 31318), UAS-upd1RNAi (BDSC: 28722), UAS-upd1OE (BDSC: 67555), UAS-Hex-ARNAi (VDRC: KK 104680), UAS-AldRNAi (BDSC: 65884), UAS-PgkRNAi (VDRC: KK 110081), UAS-PyKRNAi (VDRC: GD 49533), UAS-fbpRNAi (VDRC: KK 108554), UAS-PDHRNAi (VDRC: GD 40410), UAS-AconRNAi (BDSC: 34028), UAS-Mdh2RNAi (BDSC: 36606), UAS-Mpc1RNAi (VDRC: KK 103829), UAS-PCBRNAi (VDRC: KK 105936), UAS-kdnRNAi (BDSC: 36740), UAS-ATPCLRNAi (VDRC: GD 30282), UAS-IndyRNAi (VDRC: GD 9982), UAS-tra2RNAi (BDSC: 28018), UAS-AstA-R2RNAi (BDSC: 25935), UAS-TkR99DRNAi (BDSC: 27513), UAS-rkRNAi (BDSC: 31958), UAS-PutRNAi (BDSC: 35195), UAS-InRRNAi (BDSC: 35251), UAS-LRP1RNAi (BDSC: 44579), UAS-baboRNAi (BDSC: 25933), UAS-LpR1RNAi (BDSC: 50737), UAS-mglRNAi (BDSC: 33940), UAS-LpR2RNAi (BDSC: 54461), UAS-torRNAi (BDSC: 35639), UAS-ITPRNAi (BDSC: 25799), UAS-TlRNAi (BDSC: 35628), UAS-MlxRNAi (VDRC: KK 110630), UAS-MondoRNAi (VDRC: KK 109821), UAS-MetRNAi (VDRC: KK 100638), UAS-Npc2eRNAi (VDRC: KK 100445), UAS-NLazRNAi (VDRC: KK 107553), UAS-grndRNAi (VDRC: KK 104538), UAS-slifRNAi (VDRC: GD 45590), UAS-AdipoRRNAi (VDRC: GD 40936), UAS-EcRRNAi (VDRC: GD 37058), UAS-uspRNAi (VDRC: GD 16893), UAS-btlRNAi (BDSC: 60013), UAS-gceRNAi (BDSC: 61852), UAS-Mal-A1RNAi (VDRC: KK 106220), UAS-CG6484RNAi (VDRC: KK 109484), UAS-PdpRNAi (VDRC: KK 107271), UAS-PdkRNAi (VDRC: KK 106641), UAS-CG13907RNAi (VDRC: KK 107339), UAS-Mct1RNAi (VDRC: KK 106773), UAS-PrestinRNAi (VDRC: GD 5341), UAS-OutRNAi (VDRC: GD 51157), UAS-CG8925RNAi (VDRC: KK 101128).u-700μδ6 , UAS-lacupd3-GFP , UAS-hopdomeΔCYT , UAS-dcrtraKO (BDSC: 67412), Df(3L)st-j7 (BDSC: 5416), traF K-IN , Df(2R)trix (BDSC: 1896), tra21 , snf148 (BDSC: 7398), esgSHOF Exel7027 (BDSC: 7801). esgSHOF , zpgz-25-RKO51-2 , AkhAPaand AkhA , AkhR1   1/500, and rhodamine Phalloidin  1/1000. Fluorescent secondary antibodies  were obtained from Jackson Immunoresearch. Vectashield with DAPI (Vector Labs) was used to stain DNA.The following primary antibodies were used: chicken anti-GFP  1/10000, mouse anti-GFP  1/1000, chicken anti-beta Galactosidase  1/200, rabbit anti-phospho-Histone H3 Ser10  1/500, mouse anti-Fas3  1/50, rabbit anti-Pyruvate dehydrogenase E1-alpha subunit (phospho S293)  1/200, rabbit anti-Aconitase 2  1/200, rabbit anti-cleaved Mal-A3, Pgi and Amy-p respectively: CBGtg9060D0780D, CBGtg9060F0441D and CBGtg9060B10205D. The clones were sequence-verified and transgenic lines were established through ΦC-31 integrase mediated transformation (Bestgene). attP sites used were VK33 (BDSC: 9750) for Mal-A3 and Amy-p, and attP40 (BDSC: 36304) for Pgi.The following GFP-tagged clones from the fosmid library TransgeneOme Resource . The swapping strategy previously described in . Flies with both components were then crossed to flies with both germline-expressing Cre and ΦC-31 transgenes (BDSC: 60299). Offspring were then crossed to flies carrying a UAS-GFP reporter (BDSC: 60291) and the progeny were screened by fluorescence microscopy. Recombinants were selected to establish stable lines.To generate a knock-in ribed in  was empltra knock-in allele, the tra locus (3869 nucleotides (nt) containing: tra coding region, the 1910 nt upstream and the 967 nt downstream) was cloned using the following primer pair: 5′-AAAACGGCCGGACAGCACAACCAGTTCCGAC-3′ and 5′-AAAACTCGAGATGCCCATCCCCTGCAATAC-3′. PCR was performed with Q5 high-fidelity polymerase from New England Biolabs (M0491S). The PCR product was digested with EagI and XhoI prior to cloning into the RIV FRTnMCS1FRT white vector . The generated allele rescue tra null mutant females to fertility.To generate an excisable FRT-flanked rated by . The con by  was cloned using the following primer pair: 5′-AAAAGAATTCAATTTGTTTTATTTGTGCCTG-3′ and 5′-AAAACTCGAGAGTTTCGTCCGCGGGTC-3′. PCR was performed with Q5 high-fidelity polymerase from New England Biolabs (M0491S). The PCR product was digested with EcoRI and XhoI prior to cloning into the RIV FRTnMCS1FRT white vector , Amy-p (gRNA: TCTACAACATGGTGGCCTTCCGG) and Amy-d (gRNA: TCTACAACATGGTGGCCTTCCGG) genes, the three gRNAs were assembled from two overlapping PCR products. PCRs were performed with Q5 high-fidelity polymerase from New England Biolabs (M0491S). The final PCR product was then cloned into Bbs1 digested pCFD6 vector . The expression efficiency of the recovered transformant lines was assessed by crossing them to a mushroom body GAL4 driver. The line used in this study was the one found to have the highest expression and harbors an insertion into chromosome II.The pcDNA3.1(-)Laconic plasmid  was digeKlebsiella pneumoniae histidine sensor kinase CitA (amino acids: 6-130), inserted between the FRET pair Venus/CFP; CIT96 carries a point mutation (K77A) decreasing the affinity for citrate and CIT0 is a control sensor which harbors a mutation (R66A) that completely abolishes citrate binding. These three sensors were cloned into the pUASTattb vector (PMID: 17360644) with EcoRI and NotI. The constructs were sequence-verified and transgenic lines were established through ΦC-31 integrase mediated transformation .Three different nanosensors for citrate were generated by gene synthesis (GenScript): CIT96, CIT8 and CIT0 . CIT8 coVha100-4, which is not sexually dimorphic. For data display purposes, the average of the expression abundance was arbitrarily set at 100% for each gene for control males, and percentage of that expression is displayed for all sexes and genotypes. In graphs displaying expression of the five gut-specific sugar genes , the median of expression of these five genes taken together is also displayed for both sexes. See Table S3 for primer details such as sequences and efficiency.RNAs were extracted from 20 whole flies using Trizol (Invitrogen). RNAs were cleaned using RNAeasy mini Kit (QIAGEN), and cDNAs were synthesized using the iScript cDNA synthesis kit (Bio-Rad) from 500 ng of total RNAs. Quantitative PCRs were performed by mixing cDNA samples (5 ng) with iTaq Universal SYBR® Green Supermix  and the relevant primers in 384-well plates. Expression abundance was calculated using a standard curve for each gene, and normalized to the expression of tra mutant females used for GSE74775. A summary of relevant data for the intestinal sugar genes is provided in Table S1.The RNA-seq transcriptional data of adult midguts obtained from virgin males, females, and 2, 4 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, 5 mM trehalose, 5 mM HEPES; pH 7.1; around 350 mOsm). The dissected organs were placed into an open μ-slide  and analyzed using a confocal microscope. Fluorescent images were acquired using a 20x objective, and the following filter sets: excitation 405 nm, emission 470-522 nm (CFP channel); excitation 405 nm, emission 532-627 nm (FRET channel). For data analysis, regions of interest (ROI) were delimited and the average intensity of both mTFP and Venus channels over each ROI were calculated. The design of the laconic sensor is such that FRET from mTFP to Venus decreases when lactate concentration increases. To obtain a signal that positively correlates with lactate concentration, the inverse FRET ratio was calculated by dividing mTFP intensity by Venus intensity. For experiments with the FLII12Pglu-700μδ6 glucose sensor or the citrate sensors, the FRET ratio (YFP/CFP) was computed to obtain a signal positively correlated to glucose or citrate concentrations. For the experiments displayed in All imaging experiments were performed on dissected midguts or testes expressing laconic, the glucose sensor or the citrate sensors. Adult midguts or testes of 5-day-old flies were dissected in HL3 buffer . Samples for GC-MS analysis were processed as previously described   in pyridine, and incubated for 1h at 30°C. 25 μL of this solution were added to auto sampler vials. 60μL of N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA) were added automatically via the auto sampler and incubated for 30 min at 37°C with shaking. After incubation, 1 μL of the prepared sample was injected into the gas chromatograph inlet in the split mode with the inlet temperature held at 250°C. A 10:1 split ratio was used for analysis. The gas chromatograph had an initial temperature of 60°C for 1 min, followed by a 10°C/min ramp to 325°C and a hold time of 2 min. A 30 m Agilent Zorbax DB-5MS with 10 m Duraguard capillary column was employed for chromatographic separation. Helium was used as the carrier gas at a rate of 1 mL/min. Data was collected using MassHunter software (Agilent). Metabolites were identified and their peak area was recorded using MassHunter Quant. This data was transferred to an Excel spread sheet . Metabolite identity was established using a combination of an in-house metabolite library developed using pure purchased standards, the NIST library and the Fiehn library. Data was normalized to both the sample mass and an internal standard (d27-myristic acid). Statistical analysis was performed using Metaboanalsyt 3.0 (yst.ca/) .13C3 citric acid 99% - Cambridge Isotope Laboratories, USA) was added as an internal standard in the extraction solution (150 ng/mL). Following vortex mixing (30 s) and sonication on an ultrasonic water bath (10 min), samples were centrifuged . Finally, the supernatants were collected, filtered using PTFE membrane (0.22 μm) and transferred to autosampler vials prior to injection on the liquid chromatography system. Total protein content was determined from the pellet obtained after centrifugation  by agitation in RIPA buffer , centrifugation  and measurement of protein content using a BCA assay kit .For hemolymph extractions, males were decapitated in groups of 15-20 and placed in a 0.5 ml Eppendorf tube perforated with a 30G needle. These Eppendorf tubes were placed inside 1.5 ml Eppendorf tubes and were centrifuged for 15 min at 1500 g at 4°C to collect their hemolymph as described in . For eacChromatographic analyses were carried out on a Vanquish Flex Binary UHPLC system  coupled to a benchtop hybrid quadrupole-Orbitrap Q Exactive mass spectrometer . Baseline separation of isocitric acid and citric acid was achieved using a C18 Accucore Thermo Scientific column  equipped with vanguard column , both held at a temperature of 40°C and a flow rate of 0.2 mL/min. Mobile phases were water with 0.5% formic acid (v/v) (Solvent A) and 90% acetonitrile with 0.5% formic acid (v/v) (Solvent B). The gradient elution was performed with a 0%–80% solvent B gradient over 5 min, followed by column washing and equilibration, yielding a total run time of 13 min. Ionization was performed in the negative ion mode using a heated electrospray ionization source (HESI), under the following conditions: spray voltage −3.0 KV, heater temperature 330°C, capillary temperature 320°C, S-lens RF level 50, sheath and auxiliary gas flow rate, 35 and 10 units, respectively. Mass accuracy was calibrated using a customised calibration solution prior to sample analysis. Data was acquired in profile mode using Parallel Reaction Monitoring (PRM) with information regarding all the compounds defined in the inclusion list (see Table below), at a MS2 resolution of 17,500 at m/z 200 and isolation window of m/z 2.0. Nitrogen was used as collision gas in the higher energy collision dissociation (HCD) cell with normalized collision energy (NCE) set to 10%. Automatic gain control (AGC) was set to 2e4 and maximum injection time 50 ms. Xcalibur version 4.1 was used for data acquisition and processing.Hemolymph was prepared as above. Each sample consisted of pooled hemolymph of a total of 120-280 mated males , which was diluted 1:6 in distilled water prior to metabolomics analysis. Metabolome analysis was performed in 4 samples of fly adult body fluid per genotype using CE-TOFMS by Human Metabolome Technologies, Inc (HMT). Each sample was mixed with 450 μL of methanol containing internal standards (20 μM) and mixed. Then, chloroform (500 μL) and Milli-Q water (200 μL) were added, mixed thoroughly and centrifuged . The water layer (400 μL) was filtrated through a 5kDa cut-off filter  to remove macromolecules. The filtrate was centrifugally concentrated and resuspended in 50 μL of ultrapure water immediately before the measurement. The compounds were measured in the Cation and Anion modes of CE-TOFMS based metabolome as previously described . All CE−CE−MS Conditions for Cationic Metabolites. Separations were carried out on a fused silica capillary  using 1M formic acid as the electrolyte. Sample was injected with a pressure injection of 50 mbar for 3 s (3 nL). The applied voltage was set at +30kV. The capillary temperature was maintained at 20°C using a thermostat and the sample tray was cooled below 5°C. 5 mM ammonium acetate in 50% (v/v) methanol−water was delivered as the sheath liquid at 10 μL/min. ESI−MS was conducted in the positive ion mode and the capillary voltage was set at 4000V. A flow of heated dry nitrogen gas (heater temperature of 300°C) was maintained at 10 L/min. In MS with selective ion monitoring (SIM), sets of 30 protonated [M+H]+ ions were analyzed successively to cover the whole range of m/z values from 70 through 1027.CE−MS Conditions for Anionic Metabolites. A cationic polymer coated SMILE (+) capillary was obtained from Nacalai Tesque  and used as the separation capillary . The electrolyte for the CE separation was 50 mM ammonium acetate solution, pH 8.5. Sample was injected with a pressure injection of 50mbar for 30 s (30nL). The applied voltage was set at −30 kV. ESI−MS was conducted in the negative ion mode and the capillary voltage was set at 3500 V. In MS with SIM, sets of 30 deprotonated [M−H]- ions were analyzed successively to cover the whole range of m/z values from 70 through 1027. Other conditions were the same as in cationic metabolite analysis.Peaks detected in CE-TOFMS analysis were extracted using automatic integration software (MasterHands ver. 2.17.1.11 developed at Keio University) in order to obtain peak information including m/z, migration time (MT), and peak area. Putative metabolites were then assigned from HMT’s standard library and Known-Unknown peak library on the basis of m/z and MT. All the metabolite concentrations were calculated by normalizing the peak area of each metabolite with respect to the area of the internal standard and by using standard curves, which were obtained by single-point (100 μM or 50 μM) calibrations. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were performed by statistical analysis software (developed at HMT).Mitotic and meiotic indices were quantified by counting pH3-positive cells in > 40 testes or > 10 midguts per genotype and/or condition .For quantification of intestinal GFP protein expression level, a midgut portion (corresponding to R2 or R4 regions) was imaged at 20x magnification. GFP level was quantified using ImageJ in areas of identical size across all genotypes. Threshold was adjusted for the GFP channel (ImageJ function: Image > Adjust > Threshold) to subtract background, then the size and the intensity mean of the area above the threshold was considered . Data was collected from at least 10 midguts per genotype and/or sex, and is displayed as boxplots showing all data points.For quantification of testis GFP protein expression patterns, whole testes were imaged at 20x magnification. GFP area was quantified using ImageJ. Threshold was adjusted for the GFP channel (ImageJ function: Image > Adjust > Threshold) to subtract background, then the size of the area above the threshold was considered  and averaged by testis size. Data was collected from at least 25 testes per genotype, and is displayed as boxplots showing all data points.Left wings of females and males were dissected, dehydrated in ethanol and mounted between slide and coverslip in Euparal mounting medium. Slides were dried on a heating block overnight (60°C). Wing areas were quantified using ImageJ by manually selecting the Cartesian coordinates of six landmarks that represent junctions of veins with the wing contour, and then measuring the number of pixels included in the resulting outline . Asterisks highlighting significant comparisons across sexes are displayed in gray boxes, whereas those highlighting significant comparisons within same-sex datasets are displayed in red boxes  and blue boxes .All statistical analyses were carried out in GraphPad Prism 7.04. Comparisons between two genotypes and/or conditions were analyzed with the Mann-Whitney-Wilcoxon rank sum test. The Mann-Whitney-Wilcoxon rank sum test does not require the assumption of normal distributions, so no methods were used to determine whether the data met such assumptions. All graphs were generated using GraphPad Prism 7.04. All confocal and bright field images belonging to the same experiment and displayed together in our figures were acquired using the exact same settings. For visualization purposes, level, and channel adjustments were applied using ImageJ to the confocal images shown in the figure panels , but all quantitative analyses were carried out on unadjusted raw images or maximum projections. In all figures, n denotes the number of midguts, wings, testes, flies or group of flies that were analyzed for each genotype. Data are presented as boxplots with all data points shown, p values from Mann-Whitney-Wilcoxon test (non-significant (ns): p > 0.05; GSE74775.The accession number for gene expression reported in this paper is GEO: Data in this paper are available upon request to the Lead Contact.


Drosophila testis, exhibit age-associated decline in number and activities, yet underlying mechanisms are not fully understood. Here we show that Lin28, a highly conserved RNA binding protein, is expressed in hub cells and its expression dramatically declines in old testis. lin28 mutant testes exhibit hub cell loss and defective hub architecture, recapitulating the normal aging process. Importantly, maintained expression of Lin28 prolongs hub integrity and function in aged testes, suggesting that Lin28 decline is a driver of hub cell aging. Mechanistically, the level of unpaired (upd), a stem cell self-renewal factor, is reduced in lin28 mutant testis and Lin28 protein directly binds and stabilizes upd transcripts, in a let-7 independent manner. Altogether, our results suggest that Lin28 acts to protect upd transcripts in hub cells, and reduction of Lin28 in old testis leads to decreased upd levels, hub cell aging and loss of the stem cell niche.Age-related decline in stem cell function is observed in many tissues from invertebrates to humans. While cell intrinsic alterations impair stem cells, aging of the stem cell niche also significantly contributes to the loss of tissue homeostasis associated with reduced regenerative capacity. Hub cells, which constitute the stem cell niche in the  Stem cells, which are characterized by their ability to self-renew, present in most adult tissues produce daughter stem cells and differentiated cells. With these unique properties, stem cells replenish aged cells to maintain tissue homeostasis throughout lifespan. With age, most stem cells lose their self-renewing ability, leading to loss of stem cell number, which in turn results in tissue aging and deterioration –5. ImporDrosophila testis has been used as an incisive genetic model to provide insights into the mechanisms underlying aging processes occurring in the male germline stem cells and their niches  (#17298), were obtained from Bloomington Drosophila Stock Center. W1118 from Bloomington Drosophila Stock Center was used as a wildtype control. upd-GAL4 was a kind gift from S. Dinardo. lin28df30 was a kind gift from F. Michon. UAS-Lin28 was generated in the lab. upd-GAL4;tub-GAL80ts was a kind gift from Christian Bokel.updGal4; tubGAL80ts and updGal4;tub-GAL80ts>UAS-lin28 flies were raised at 18oC unless otherwise noted. Flies were raised at 18oC for 5 days post-eclosion to restrict GAL4 during final developmental stages. Flies were shifted to 29oC for 2 days to inhibit Gal80ts and activate GAL4 or aged for 27 days at 29oC.All To quantify the number of hub cells, the testis was stained with antibody specific to hub cell, FasIII and TO-PRO3/Hoechst a nuclear marker. Densitometric analysis of Serial Z section was used to quantify the number of hub cells using LAS X software from Leica. The number of GSCs were quantified by the cells positively stained to VASA which were directly in contact to the FasIII positive hub cells. The size of the hub cells was measured as described .lin28 cDNA (RE05342) was obtained from Drosophila Genetic Resource Centre. RE05342 consists of pFLC-1 vector which is a derivative of pBluescriptII-SK (+) with the cDNA insert (1165bp) of lin-28 (transcript 1152bp with 5’UTR and 3’UTR). The Not I-Xho I insert of the lin28 CDS (588bp) was sub-cloned into pAc5.1/V5- HisA-HA/FLAG (Invitrogen) vector for S2 cell expression, while Bgl II- Xho I insert and EcoR I-Xho I insert was sub-cloned into pUASt for fly expression studies. The KpnI-Not1 insert was sub-cloned into phmKGN/C-MC/MN vector for fragment complementation assay. The IGF-II mRNA binding protein (IMP) cDNA (RE72930) was obtained from Drosophila Genetic Resource Centre. RE72930 consists of derivative of pBluescriptII-SK (+) with cDNA insert (3955bp) of IMP-RJ (3942bp transcript with 5’UTR and 3’UTR). The Not I-Xho I insert of the IMP CDS (1743bp) was sub-cloned into pAc5.1/V5-HisA-FLAG for S2 cell related experiments.The BS-UPD) was obtained from Douglas A. Harrison. BS-UPD consists of the cDNA of upd (2520bp). The XhoI -Xba I insert of 3’UTR (740bp) of upd was sub-cloned into pAc5.1A(Invitrogen) following a luciferase reporter (pAc5.1A-Luc-upd3’UTR). Site directed mutagenesis was used to generate pAc5.1A-Luc- upd3’UTRmut (228GGAGA=AACAT).The outstretched (os)/unpaired (upd) cDNA .oC. Secondary antibodies were used at 1:500. After the secondary antibody treatment, the testis was carefully detached from the posterior region using tungsten needles and mounted in antifade. Adult testis of appropriately aged male flies were immunostained as previously described except they were hand dissected in Ringer’s solution and fixed in 4% formaldehyde for 40 minutes.Immunostaining was done as described . BrieflyThe following primary antibodies were used along with the following concentration: Rat anti-Lin28 1:100 (This study), mouse anti-FasIII 1:30, rat DE Cadherin 1:30, rat anti-VASA 1:400 , Rabbit anti-VASA 1:3000, Rabbit anti-Zfh1(1:1000) (Ruth. Lehmann), guinea pig anti-Traffic Jam 1:10000 (Dorothea Godt), rabbit anti-IMP 1:1000 , Rat anti-Cup 1:1000 (A. Nakamura), Rabbit anti-GFP 1:500 , Rabbit anti- STAT92E 1:1000 (Denise Montell). The secondary antibodies were used in 1:500 .oC. The thawed lysate was subjected to Immunoprecipitation using HA-Agarose or FLAG-Agarose beads for 4 hours at 4oC and washed 5 times in wash buffer followed by 3 times washing in wash buffer with 1M urea. The washed Agarose beads were subjected to DNAse1 treatment for 10 minutes at 37oC followed by proteinase K (10 mg/ml) treatment at 55oC for 30 minutes. The proteinase K treated beads were subjected to Phenol: Chloroform: Isoamyl alcohol extraction to extract RNA. The RNA was diluted and was subjected to reverse transcription using reverse transcriptase. The RT samples were used as template to amplify RNA bound to the flagged proteins by PCR.S2 cells transfected with HA tagged Lin28 and FLAG tagged IMP were washed twice in ice cold PBS and were subjected to RNA-IP. The washed cells were lysed using polysome lysis buffer containing protease inhibitor cocktail, RNase inhibitor and Vanadyl complexes along with DTT. The lysed cells were centrifuged at high speed for 15 minute and frozen for 30 minutes at -80Luc-upd3’UTR or mutLuc-upd3’UTR using DDAB transfection reagent (Fluka/Sigma). 72 hours after transfection cells were harvested, and total RNA was extracted with Trizol (Invitrogen). Following reverse transcription, qRT-PCR was used to measure Luc-upd3’UTR levels using upd3’UTR-specific primers with rp49 as control.When using the luciferase-based reporters, S2 cells were transfected with reporter constructs of RNA in situ hybridization was performed as previously described . Full lelet-7 stem loop primer 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACTATA-3’ and let-7, forward, 5’-GCCGCTGAGGTAGTAGGTT GTA-3’ and reverse 5’-GTGCAGGGTCCGAGG-3’, as described previously [Total RNAs were extracted from young (0-1 day old) and old (30-32 days old or specified) testis using mirVana miRNA isolation kit (Invitrogen). Total RNA (200 ng) was used for cDNA synthesis with specific eviously . Quantitupd forward, 5’-GGGAGAGAGAGAGAAATAGAGAGA-3’ upd reverse, 5’-CGGGCGTGGCGAATAATA-3’; Imp forward, 5’-GCACCCACCACAATTTACAAC-3’, Imp reverse, 5’-CTCCAGGTTGCTTGCTTACT-3’; DE-Cadherin forward, 5’-CCGTAGACACTAAGACTCGATTAAG-3’, DE-cadherin, reverse, 5’-GTGTGGCTCTTCGTTTGTTG-3’; rp49, forward, 5’-CCAGTCGGATCGATATGCTAAG-3’, rp49, reverse, 5’-CCGATGTTGGGCATCAGATA-3’; lin28, forward 5’-CAGCCAGAGTTCCACTTCAT-3’, lin28, reverse 5’-GGCCACGTTGAACCATTTG-3’.For qRT-PCR total RNA was extracted from the appropriately aged testes using RNeasy Mini Kit (Qiagen). Total RNA (500ng) was used for cDNA synthesis with oligo dT, Quantitative PCR was performed with SYBR green  on a Step One Real-Time PCR Systems from Bio-Rad according to the manufacturer’s instructions. Specific primers for qRT are the following: The intensity measurements for the samples were created in ImageJ (NIH) using RGB Profiler Macro. A rectangular selection around the hub cells and or individual cells, which were normalized with the background to get the accurate measurement. Similar settings were used for the all the measurement in the same day for the same set of experiments.All P values were calculated using a two-tailed unpaired t-test (GraphPad Prism 7) Average values are presented as mean ± s.e.m. Unless otherwise noted, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. n.s. not significant.Supplementary File