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• W2804174839 abstract "•In vivo maintenance of the long-term hematopoietic stem cell (LT-HSC) pool relies on the RNA-binding protein arsenic resistance protein 2 (Ars2).•Ars2 supports the proliferation of early hematopoietic progenitors.•Deletion of Ars2 results in thymocyte apoptosis.•Physiological effects of Ars2 are context dependent. Recent biochemical characterization of arsenic resistance protein 2 (Ars2) has established it as central in determining the fate of nascent ribonucleic acid (RNA) polymerase II (RNAPII) transcripts. Through interactions with the nuclear 5′-7-methylguanosine cap-binding complex, Ars2 promotes cotranscriptional processing coupled with nuclear export or degradation of several classes of RNAPII transcripts, allowing for gene expression programs that facilitate rapid and sustained proliferation of immortalized cells in culture. However, rapidly dividing cells in culture do not represent the physiological condition of the vast majority of cells in an adult mammal. To examine functions of Ars2 in a physiological setting, we generated inducible Ars2 knockout mice and found that deletion of Ars2 from adult mice resulted in defective hematopoiesis in bone marrow and thymus. Importantly, only some of this defect could be explained by the requirement of Ars2 for rapid proliferation, which we found to be cell-type specific in vivo. Rather, Ars2 was required for survival of developing thymocytes and for limiting differentiation of bone marrow resident long-term hematopoietic stem cells. As a result, Ars2 knockout led to rapid thymic involution and loss of the ability of mice to regenerate peripheral blood after myeloablation. These in vivo data demonstrate that Ars2 expression is important at several steps of hematopoiesis, likely because Ars2 acts on gene expression programs underlying essential cell fate decisions such as the decision to die,proliferate, or differentiate. Recent biochemical characterization of arsenic resistance protein 2 (Ars2) has established it as central in determining the fate of nascent ribonucleic acid (RNA) polymerase II (RNAPII) transcripts. Through interactions with the nuclear 5′-7-methylguanosine cap-binding complex, Ars2 promotes cotranscriptional processing coupled with nuclear export or degradation of several classes of RNAPII transcripts, allowing for gene expression programs that facilitate rapid and sustained proliferation of immortalized cells in culture. However, rapidly dividing cells in culture do not represent the physiological condition of the vast majority of cells in an adult mammal. To examine functions of Ars2 in a physiological setting, we generated inducible Ars2 knockout mice and found that deletion of Ars2 from adult mice resulted in defective hematopoiesis in bone marrow and thymus. Importantly, only some of this defect could be explained by the requirement of Ars2 for rapid proliferation, which we found to be cell-type specific in vivo. Rather, Ars2 was required for survival of developing thymocytes and for limiting differentiation of bone marrow resident long-term hematopoietic stem cells. As a result, Ars2 knockout led to rapid thymic involution and loss of the ability of mice to regenerate peripheral blood after myeloablation. These in vivo data demonstrate that Ars2 expression is important at several steps of hematopoiesis, likely because Ars2 acts on gene expression programs underlying essential cell fate decisions such as the decision to die,proliferate, or differentiate. Arsenic resistance protein 2 (Ars2) is an essential, highly conserved protein, with mammals sharing more than 98% amino acid identity [1Wilson MD Wang D Wagner R et al.ARS2 is a conserved eukaryotic gene essential for early mammalian development.Mol Cell Biol. 2008; 28: 1503-1514Crossref PubMed Scopus (39) Google Scholar]. Ars2 was initially described as a modulator of arsenic sensitivity in a cDNA screen [2Rossman TG Wang Z Expression cloning for arsenite-resistance resulted in isolation of tumor-suppressor fau cDNA: possible involvement of the ubiquitin system in arsenic carcinogenesis.Carcinogenesis. 1999; 20: 311-316Crossref PubMed Scopus (51) Google Scholar]; subsequent analysis determined that the ability of Ars2 to modulate arsenic sensitivity likely resulted from a dominant-negative effect of the partial cDNA sequence used in the screen [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. Full-length Ars2 did not impart arsenic resistance, but rather was found to be a component of the 5′-7-methylguanosine (7mG) cap binding complex (CBC) that coordinated RNA polymerase II (RNAPII) transcription with nuclear RNA processing and export [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Gruber JJ Olejniczak SH Yong J La Rocca G Dreyfuss G Thompson CB Ars2 promotes proper replication-dependent histone mRNA 3′ end formation.Mol Cell. 2012; 45: 87-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5Andersen PR Domanski M Kristiansen MS et al.The human cap-binding complex is functionally connected to the nuclear RNA exosome.Nat Struct Mol Biol. 2013; 20: 1367-1376Crossref PubMed Scopus (150) Google Scholar, 6Sabin LR Zhou R Gruber JJ et al.Ars2 regulates both miRNA-and siRNA-dependent silencing and suppresses RNA virus infection in Drosophila.Cell. 2009; 138: 340-351Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 7Visa N Izaurralde E Ferreira J Daneholt B Mattaj IW A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export.J Cell Biol. 1996; 133: 5-14Crossref PubMed Scopus (196) Google Scholar, 8Giacometti S Benbahouche NEH Domanski M et al.Mutually exclusive CBC-containing complexes contribute to RNA fate.Cell Rep. 2017; 18: 2635-2650Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 9Schulze WM Cusack S Structural basis for mutually exclusive co-transcriptional nuclear cap-binding complexes with either NELF-E or ARS2.Nat Commun. 2017; 8: 1302Crossref PubMed Scopus (18) Google Scholar]. Ars2 was required for rapid proliferation of cells in culture [2Rossman TG Wang Z Expression cloning for arsenite-resistance resulted in isolation of tumor-suppressor fau cDNA: possible involvement of the ubiquitin system in arsenic carcinogenesis.Carcinogenesis. 1999; 20: 311-316Crossref PubMed Scopus (51) Google Scholar, 3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar] and, unlike other protein components of the CBC, it could be induced by mitogenic signals [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Olejniczak SH La Rocca G Gruber JJ Thompson CB Long-lived microRNA–argonaute complexes in quiescent cells can be activated to regulate mitogenic responses.Proc Natl Acad Sci USA. 2013; 110: 157-162Crossref PubMed Scopus (50) Google Scholar]. The intimate relationship between Ars2 and cellular proliferation likely stems from its role in 3′ end processing of replication-dependent histone (RDH) mRNAs [4Gruber JJ Olejniczak SH Yong J La Rocca G Dreyfuss G Thompson CB Ars2 promotes proper replication-dependent histone mRNA 3′ end formation.Mol Cell. 2012; 45: 87-98Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11O'Sullivan C Christie J Pienaar M et al.Mutagenesis of ARS2 domains to assess possible roles in cell cycle progression and microRNA and replication-dependent histone mRNA biogenesis.Mol Cell Biol. 2015; 35: 3753-3767Crossref PubMed Scopus (14) Google Scholar, 12Hallais M Pontvianne F Andersen PR et al.CBC–ARS2 stimulates 3′-end maturation of multiple RNA families and favors cap-proximal processing.Nat Struct Mol Biol. 2013; 20: 1358-1366Crossref PubMed Scopus (104) Google Scholar]. In addition to RDH mRNAs, Ars2 was shown to be involved in maturation and/or nuclear export of microRNAs, small nuclear RNAs, small nucleolar RNAs, and select mRNAs [10Olejniczak SH La Rocca G Gruber JJ Thompson CB Long-lived microRNA–argonaute complexes in quiescent cells can be activated to regulate mitogenic responses.Proc Natl Acad Sci USA. 2013; 110: 157-162Crossref PubMed Scopus (50) Google Scholar, 11O'Sullivan C Christie J Pienaar M et al.Mutagenesis of ARS2 domains to assess possible roles in cell cycle progression and microRNA and replication-dependent histone mRNA biogenesis.Mol Cell Biol. 2015; 35: 3753-3767Crossref PubMed Scopus (14) Google Scholar, 13Laubinger S Sachsenberg T Zeller G et al.Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana.Proc Natl Acad Sci USA. 2008; 105: 8795-8800Crossref PubMed Scopus (285) Google Scholar, 14Vasudevan S Tong Y Steitz JA Switching from repression to activation: microRNAs can up-regulate translation.Science. 2007; 318: 1931-1934Crossref PubMed Scopus (2153) Google Scholar, 15Flaherty SM Fortes P Izaurralde E Mattaj IW Gilmartin GM Participation of the nuclear cap binding complex in pre-mRNA 3′ processing.Proc Natl Acad Sci USA. 1997; 94: 11893-11898Crossref PubMed Scopus (172) Google Scholar]. As a balance to its anabolic effects on RNAPII transcripts, Ars2 supports transcription termination-coupled degradation of superfluous transcripts such as promoter upstream transcripts and enhancer RNAs [5Andersen PR Domanski M Kristiansen MS et al.The human cap-binding complex is functionally connected to the nuclear RNA exosome.Nat Struct Mol Biol. 2013; 20: 1367-1376Crossref PubMed Scopus (150) Google Scholar, 16Iasillo C Schmid M Yahia Y et al.ARS2 is a general suppressor of pervasive transcription.Nucleic Acids Res. 2017; 45: 10229-10241Crossref PubMed Scopus (36) Google Scholar, 17Lubas M Christensen MS Kristiansen MS et al.Interaction profiling identifies the human nuclear exosome targeting complex.Mol Cell. 2011; 43: 624-637Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar]. Overall, our data indicate that the net effect of Ars2 is increased efficiency of productive RNAPII transcription.In vivo characterization of Ars2 in mammals, although limited, demonstrated roles for Ars2 that do not stem from its requirement for rapid cellular proliferation. Wilson et al. demonstrated that embryos at the blastocyst stage rely on Ars2 to promote cell survival [1Wilson MD Wang D Wagner R et al.ARS2 is a conserved eukaryotic gene essential for early mammalian development.Mol Cell Biol. 2008; 28: 1503-1514Crossref PubMed Scopus (39) Google Scholar], whereas Gruber et al. found similar viability-promoting properties of Ars2 in adult peripheral immune organs [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. Finally, Andreu-Agullo et al. demonstrated that Ars2 is essential for maintenance of neural stem cells in the absence of effects on their proliferation or viability [18Andreu-Agullo C Maurin T Thompson CB Lai EC Ars2 maintains neural stem-cell identity through direct transcriptional activation of Sox2.Nature. 2012; 481: 195-198Crossref Scopus (61) Google Scholar]. Rather, Ars2 activated transcription of Sox2, a transcription factor that suppresses differentiation of neural stem cells.In the current study, we set out to identify additional physiologic roles for Ars2 in adult mammals. To this end, we generated inducible whole-body Ars2 knockout (Ars2 iKO) mice by crossing Ars2-floxed mice [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar] with mice expressing the Cre-ERT2 transgene [19Feil S Valtcheva N Feil R Inducible cre mice.Methods Mol Biol. 2009; 530: 343-363Crossref PubMed Scopus (163) Google Scholar]. Initial characterization of Ars2 iKO mice after induced whole-body Ars2 deletion confirmed that developing blood cells are sensitive to Ars2 deletion, as originally observed by Gruber et al. after MxCre-mediated Ars2 deletion [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. Adult mammals continually regenerate blood cells in a well-characterized, stepwise process termed hematopoiesis. Hematopoiesis begins in the bone marrow with multipotent long-term hematopoietic stem cells (LT-HSCs) that are capable of indefinite self-renewal and differentiation to generate all blood cells throughout the life of an organism [20Babovic S Eaves CJ Hierarchical organization of fetal and adult hematopoietic stem cells.Exp Cell Res. 2014; 329: 185-191Crossref PubMed Scopus (59) Google Scholar, 21Seita J Weissman IL Hematopoietic stem cell: self‐renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (493) Google Scholar]. In adult mammals, the majority of LT-HSCs arequiescent to prevent exhaustion and malignant transformation [22Cheshier SH Morrison SJ Liao X Weissman IL In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells.Proc Natl Acad Sci USA. 1999; 96: 3120-3125Crossref PubMed Scopus (662) Google Scholar, 23Wilson A Laurenti E Oser G et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1352) Google Scholar]. Upon activation by peripheral signaling, LT-HSCs enter the cell cycle and undergo asymmetrical division to self-renew and differentiate into downstream lineage committed stem and progenitor cells that ultimately produce every mature blood cell [23Wilson A Laurenti E Oser G et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1352) Google Scholar, 24Cabezas-Wallscheid N Buettner F Sommerkamp P et al.Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy.Cell. 2017; 169 (e19): 807-823Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 25Zon LI Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal.Nature. 2008; 453: 306-313Crossref PubMed Scopus (225) Google Scholar].Although many blood cells mature before leaving the bone marrow, most T cells require additional, antigen-dependent maturation that takes place in the thymus. Bone-marrow-derived T-cell progenitors enter the thymus and progress through a series of maturation and selection processes termed thymopoiesis that ultimately yields mature CD4+ helper T cells and CD8+ cytotoxic T cells [26Krueger A Ziętara N Łyszkiewicz M T cell development by the numbers.Trends Immunol. 2017; 38: 128-139Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Witt CM Robey EA Thymopoiesis in 4 dimensions.Semin Immunol. 2005; 17: 95-102Crossref PubMed Scopus (12) Google Scholar, 28Schwarz BA Bhandoola A Trafficking from the bone marrow to the thymus: a prerequisite for thymopoiesis.Immunol Rev. 2006; 209: 47-57Crossref PubMed Scopus (63) Google Scholar]. Early thymic maturation during the CD4/CD8 double-negative (DN) stage of thymopoiesis is driven by recombination of T-cell receptor (TCR) genes to produce TCR diversity [29Ueda-Hayakawa I Mahlios J Zhuang Y Id3 restricts the developmental potential of γδ lineage during thymopoiesis.J Immunol. 2009; 182: 5306-5316Crossref PubMed Scopus (70) Google Scholar]. After TCR recombination, thymopoiesis proceeds to the CD4/CD8 double-positive (DP) stage, during which positive and negative selection eliminate non-major-histocompatibility complex-restricted and autoreactive T-cell progenitors. The small fraction of T-cell progenitors that survive positive and negative selection downregulate either CD4 or CD8 to become mature CD8+ or CD4+ T cells, respectively [30Starr TK Jameson SC Hogquist KA Positive and negative selection of T cells.Annu Rev Immunol. 2003; 21: 139-176Crossref PubMed Scopus (1175) Google Scholar].The current study extends cursory observations regarding roles for Ars2 in normal mammalian hematopoiesis made by Gruber et al. [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. Specifically, we show that thymopoiesis is critically dependent on expression of Ars2, both in developing thymocytes and in thymic stroma. In addition, we report that hematopoiesis within the bone marrow of Ars2-deficient mice is compromised as the result of defective proliferation of progenitor cells combined with loss of self-renewal capacity of LT-HSCs. Taken together, our data indicate that Ars2 is essential for multiple processes involved in production of mature blood cells and influences these processes through intrinsic effects on developing blood cells along with effects on stromal cells that support hematopoiesis.MethodsMiceMice were maintained in Laboratory Animal Shared Resource at Roswell Park Cancer Institute and all procedures were approved by the institutional animal care and use committee (IACUC). Four different strains of mice were used in this study: C57BL/6J (B6), Stock No. 000664, B6.Cg-Tg(UBC-Cre/ERT2)1Ejb/1J (Cre-ERT2), Stock No. 007001 and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (mT/mG), Stock No. 007576 were purchased from The Jackson Laboratory. Ars2 floxed (Ars2fl/fl) mice were obtained from the Thompson laboratory [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar]. Cre-ERT2 Ars2fl/fl, mT/mG Ars2fl/fl, and mT/mG Cre-ERT2-Ars2fl/fl were generated by crossbreeding of Cre-ERT2 and mT/mG with Ars2fl/fl mice. To delete Ars2 gene from its floxed sites, Cre-ERT2 was activated by intraperitoneal injection of 75μg of tamoxifen (Sigma-Aldrich, 10540-29-1) per gram bodyweight in 6- to 8-week-old mice for 5 days. Chimera mice were generated by tail vein injection of 2.5–5million bone marrow cells from Ars2fl/fl or Cre-ERT2 Ars2fl/fl or mT/mG Ars2fl/fl or mT/mG Cre-ERT2-Ars2fl/fl into lethally irradiated (600 Rad) C57BL/6J6 mice. Mixed chimeras were generated by tail vein injection of 1:1 ratio of bone marrow cells from both Ars2fl/fl and Cre-ERT2-Ars2fl/fl or mT/mG Ars2fl/fl and mT/mG Cre-ERT2-Ars2fl/fl into lethally irradiated C57BL/6J mice.Flow cytometryTo identify the stem/progenitor cell populations, bone marrow was harvested by flushing the hind limb bones using FACS buffer (1% bovine serum albumin and 0.5% sodium azide in 1 × phosphate-buffered saline [PBS]) and filtered through a 40 μmol/L cell strainer. Red blood cells were lysed using ACK lysing buffer (Thermo Fisher Scientific). Cells were stained in a two-step manner with CD34 as the first step and the rest of the antibodies as the second step. Data were collected and analyzed with a BD Biosciences LSR II A flow cytometer and FlowJo version 10 software, respectively. For cell cycle analysis, cells were stained with surface markers, fixed, permeabilized (Intrasure Kit), and stained with 4[prime],6-diamidino-2-phenylindole (DAPI). To analyze T cells, harvested thymus was meshed through a 40 µmol/L cell strainer and stained for flow cytometry. Markers used in this study are listed in Supplementary Table E1 (online only, available at www.exphem.org).Supplementary Table E1Flow Cytometry MarkersNameFlurophoreCompanyCat#NameFlurophoreCompanyCat#Lineage markersFITC or biotinBiolegendB220FITCBiolegend103206CD3FITC or biotinBiolegend100203 or 100243CD11bFITCBiolegend10106CD11BFITC or biotinBiolegend101205 or 101203NK1.1FITCBiolegend108705CD45RFITC or biotinBiolegend103305 or 103203LY6-GFITCBiolegend127605LY6GFITC or biotinBiolegend127605 or 127603CD4PEBD Pharmigen561837TER119FITC or biotinBiolegend116205 or 116203CD4PE-CY7eBioscience25-0041-81CD8PE-CY7eBioscience25-0081-81Ly6A (Sca)BV421Biolegend108127CD8APCBD Pharmigen553035CD117(c-Kit)APC-CY7BD BioscienceTCRbBV510Biolegend109233CD34APCBiolegend128611TCRbPE-DAZZLEBiolegend109239CD48BV510Biolegend103443CD25PerCPBiolegend102027CD16/32Alexa700BiolegendCD25PerCPCy5.5Biolegend1911CD150PerCP Cy5.5Biolegend115921CD44Ax700Biolegend103025CD127(IL7ra)PE Cy7 or BV650Biolegend135013 or 135043CD44PEBiolegend103007CD135 (Flt3)PEBiolegend135305 orCD24BV421BD horizon562563CD135 (Flt3)Alexa700Novus BiologicalsNBP1-43352AF700CD3FITCBiolegend100204Ki67Alexa700Biolegend652419CD3Pacific BlueBiolegend100214Annexin VBiotinBiolegend640904B220APC-CY7BD Pharmigen552094DAPIBiolegend422801Annexin VAPCBiolegend640919streptavidinBV395BD Biosience564176streptavidinBV711Biolegend405241Primary Anti caspase 3RbAbcamab13847Secondary Alexa 488Goat α RbAbcamab150077 Open table in a new tab ImmunohistochemistryFemurs were formalin fixed, decalcified, and 10μm sectioned by the pathology core facility of Roswell Park Cancer Institute. Sections were deparaffinized using the xylene and ethanol method [31Zhang G, et al. Deparaffinization compositions and methods for their use. 2003, Google Patents.Google Scholar], antigen retrieved (heat-induced antigen retrieval using Reveal Decloaker, Biocare), permeabilized by Triton 0.5%, and blocked by 5% normal goat serum in PBS. Slides were incubated with primary antibody (anti-caspase 3 [Rb], 1:200) overnight. The next day, slides were washed (1 × PBS) and incubated with secondary antibody (goat anti-rabbit Alexa Fluor 488, 1:2000) for 2hours. Finally, slides were washed (1 × PBS) and DAPI mounted. Images were taken by fluorescence microscope. Antibodies are listed in Supplementary Table E1 (online only, available at www.exphem.org).Self-renewal (5-fluorouracil) assaysAt day 7 after tamoxifen injection, 5-fluorouracil (5-FU) was administered intraperitoneally at either four weekly 120mg/kg doses or a single 150mg/kg dose. Blood was drawn from the submandibular vein and collected in 50 μmol/L ethylenediaminetetraacetic acid tubes. Complete blood count was performed using HemaTrue. Moribund mice (identified by ruffled fur, hunched back, and more than 20% reduction in weight) were euthanized according to IACUC protocol and bone marrow was harvested for flow cytometry.Colony formation assaySingle-cell colony formation assay (CFA) was performed according to a protocol described previously by Ema et al. [32Ema H Morita Y Yamazaki S et al.Adult mouse hematopoietic stem cells: purification and single-cell assays.Nat Protoc. 2006; 1: 2979-2987Crossref PubMed Scopus (144) Google Scholar] from single-sorted Lin–Sca+kit+CD34–CD150+CD48– LT-HSCs. After 14days of culture, colonies were imaged and analyzed with ImageJ. Colonies were cytospined and stained for Hemacolor according to the manufacturer's protocol (EMD Millipore).Quantitative real-time polymerase chain reactionRNA from harvested organs was extracted using TRIzol and >the miRNeasy kit (Qiagen). cDNA was generated using Superscript IV (Thermo Fisher Scientific) and quantitative real-timepolymerase chain reaction (qRT-PCR) with SYBR green master mix (Thermo Fisher Scientific) was performed using Quant Studio 6 flex device. Primers are listed in Supplementary Table E2 (online only, available at www.exphem.org). Expression of Ars2 was normalized by TBP using the ∆CT method as follows: expression level = 2(CTTBP – CTArs2).Supplementary Table E2qRT-PCR primersm TBP -FAAGGGAGAATCATGGACCAGm TBP-RCCGTAAGGCATCATTGGACTm Ars2-FGCTCACAAAGACGAGGAGTGm Ars2-RAGGGACAGGAACACCTTCAG Open table in a new tab Statistical analysisAll values are expressed as means ± standard deviation or standard error of the mean. Unpaired Student t test was used to compare two experimental groups using GraphPad Prism 7 software. p < 0.05 was considered significant.ResultsDeletion of Ars2 is lethal to adult miceAlthough growing evidence has established Ars2 as a vital factor for transcription regulation and nuclear RNA processing, little is known about its relevance to normal mammalian physiology. To begin to explore physiological relevance of Ars2 in mammals, we generated Ars2 iKO mice by cross breeding Ars2fl/fl mice [3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar] with mice expressing the tamoxifen-inducible Cre recombinase Cre-ERT2 [19Feil S Valtcheva N Feil R Inducible cre mice.Methods Mol Biol. 2009; 530: 343-363Crossref PubMed Scopus (163) Google Scholar]. In these mice, exons 2–20 of the Srrt gene, which codes for Ars2, are deleted by daily intraperitoneal injection of tamoxifen for 5 consecutive days (Figure 1A). Deletion of Srrt after tamoxifen injection was confirmed by endpoint PCR in multiple organs (Supplementary Figure E1A, online only, available at www.exphem.org) and changes in Ars2 mRNA expression was determined by qRT-PCR. Consistent with previous observations [1Wilson MD Wang D Wagner R et al.ARS2 is a conserved eukaryotic gene essential for early mammalian development.Mol Cell Biol. 2008; 28: 1503-1514Crossref PubMed Scopus (39) Google Scholar, 3Gruber JJ Zatechka DS Sabin LR et al.Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.Cell. 2009; 138: 328-339Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar], Ars2 mRNA expression was high in hematopoietic tissues of control mice. Reduced expression of Ars2 mRNA was observed in most organs 8days after first tamoxifen injection of Ars2 iKO mice (Figure 1B). Several organs from Ars2 iKO mice failed to demonstrate reduction in Ars2 mRNA expression despite clear evidence of Srrt deletion (Supplementary Figure E1A, online only, available at www.exphem.org), suggesting extended half-life of Ars2 mRNA in these organs. Importantly, tamoxifen treatment of Ars2 iKO mice led to rapid weight loss (Figure 1C) and death (Figure 1D) within 2 weeks, with no differences observed between male and female mice (Supplementary Figure E1B, online only, available at www.exphem.org). In contrast, Ars2fl/fl mice injected with tamoxifen survived for the duration of each experiment (20–60 days) and showed no signs of distress (Figures 1C and 1D). Ars2 iKO mice injected with vehicle control corn oil and Ars2fl/WT mice injected with tamoxifen also demonstrated normal survival times (data not shown).Ars2 is required for normal thymopoiesisNecropsy of tamoxifen-treated Ars2 iKO mice revealed no obvious cause of death, but a marked reduction in the size of Ars2-deleted thymi compared with littermate control mice (Figure 2A). The reduction in size was reflected in the significant reduction in cellularity and mass (Figure 2B) of thymi from tamoxifen-treated Ars2 iKO mice compared with littermate controls. In contrast, other parenchymal and immune organs such as kidney, liver, and spleen appeared normal on gross examination (Supplementary Figures E2A and E2B, online only, available at www.exphem.org).Figure2Ars2 supports survival of immature thymocytes. (A) Gross anatomy of thymi showing smaller size after tamoxifen-induced Ars2 depletion. (B) Cellularity (left) and mass (right) of thymi from 6- to 8-week-old Ars2 iKO (n = 8) mice was reduced compared with littermate controls (n = 8) 7–10days after initial tamoxifen injection (**p = 0.001 and *p = 0.01, respectively). Dots represent individual mice with lines connecting littermates. (C) Representative dot plots showing distribution of lin– (B220–, CD11b–, Ly-6G–, NK1.1–) thymocytes between four major developmental stages based on CD4 and CD8 staining. Numbers represent mean ± standard error for each of four thymocyte populations, CD4/CD8 DN (p = 0.002), CD4/CD8 DP (p = 0.007), CD4SP (p = 0.003), and CD8SP (p = 0.04) comparing Ars2 iKO (n = 8) versus control (n = 8) mice 7–8days after initial tamoxifen injection. (D) Average number ± standard deviation of DN, CD8ISP (TCRβ–, CD24+), DP, semi-mature (TCRβ+, CD24+) CD4SP and CD8SP and mature (TCRβ+, CD24–) CD4, and CD8SP thymocytes isolated from Ars2 iKO (n = 8) versus control (n = 8) mice 7–8days after initial tamoxifen injection (NS: p > 0.05, *p ≤ 0.05, **p ≤ 0.01). (E) Schematics of mixed mT/mG chimera model. (F) Percentage apoptosis (Annexin V+) ± standard deviation of immature, semi-mature, and mature thymocytes from mixed mT/mG chimeric mice (n = 4, *p = 0.01, ***p < 0.0008).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess whether thymocyte populations were altered by Ars2 depletion, multicolor flow cytometry was performed [33Hu Q Nicol SA Suen AY Baldwin TA Examination of thymic positive and negative selection by flow cytometry.J Vis Exp. 2012; 68: 4269Google Scholar]. A significant decrease in the percentage of CD" @default.
• W2804174839 created "2018-06-01" @default.
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• W2804174839 creator A5042261060 @default.
• W2804174839 creator A5078962454 @default.
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• W2804174839 date "2018-08-01" @default.
• W2804174839 modified "2023-10-15" @default.
• W2804174839 title "The RNA binding protein Ars2 supports hematopoiesis at multiple levels" @default.
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