A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency

A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency Vincent Giudice , Florence Perold , Yannicke Pijoff , Nathalie Doerflinger , Nicolas Allègre , Claire Chazaud , Irène Aksoy , Pierre Savatier , Pierre-Yves Bourillot doi: https://doi.org/10.1101/2025.09.23.677977 Vincent Giudice 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Florence Perold 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yannicke Pijoff 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nathalie Doerflinger 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicolas Allègre 2 Université Clermont Auvergne, CNRS, INSERM, GReD Institute, Faculté de Médecine , F-63000, Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Claire Chazaud 2 Université Clermont Auvergne, CNRS, INSERM, GReD Institute, Faculté de Médecine , F-63000, Clermont-Ferrand, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Irène Aksoy 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pierre Savatier 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: pierre-yves.bourillot{at}inserm.fr Pierre-Yves Bourillot 1 Univ Lyon, Université Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208 , INRAE USC 1361, F-69500 Bron, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: pierre-yves.bourillot{at}inserm.fr Abstract Full Text Info/History Metrics Preview PDF Summary Leukemia inhibitory factor (LIF)/STAT3 signaling is central to maintaining naïve pluripotency in mouse embryonic stem cells (mESCs). We identify Asgard , a previously uncharacterized long non-coding RNA, as a direct STAT3 target required for efficient self-renewal. Asgard is rapidly induced by LIF, enriched in the epiblast, and its depletion reduces alkaline phosphatase–positive colony formation while enhancing differentiation. Mechanistically, Asgard fulfils a dual role: it acts as the primary transcript for the differentiation-promoting microRNA Odin , while also functioning as a sponge to sequester Odin and related miRNAs. This dual mechanism enables Asgard to both generate and buffer pro-differentiation signals, thereby stabilizing the pluripotent state while preserving responsiveness to lineage cues. Our work reveals a new paradigm in RNA-mediated control of stem cell identity, where a single STAT3-regulated lncRNA couples microRNA production with competitive inhibition to safeguard naïve pluripotency. Introduction Mouse embryonic stem cells (mESCs) provide a robust system to dissect the molecular logic of naïve pluripotency. Their self-renewal is maintained by cytokine signaling through the leukemia inhibitory factor (LIF)/STAT3 pathway, which activates a transcriptional program centered on key regulators such as Klf4 , Tfcp2l1 , and Esrrb (Aksoy et al , 2014; Martello et al , 2013; Matsuda et al , 1999; Niwa et al , 2009). Constitutive activation of STAT3 is sufficient to sustain naïve pluripotency, underscoring its role as a master transcriptional regulator (van Oosten et al , 2012). In addition to transcription factors, non-coding RNAs (ncRNAs) have emerged as pivotal regulators of stem cell fate. Among these, microRNAs (miRNAs) form integral components of the pluripotency network (Wang et al , 2007). For example, the miR-290–295 cluster reinforces self-renewal by targeting cell-cycle inhibitors and differentiation cues, while let-7 family members promote exit from pluripotency and lineage commitment (Judson et al , 2009; Melton et al , 2010). The dynamic balance between pluripotency-supporting and differentiation-promoting miRNAs exemplifies how small RNAs act as molecular switches controlling stem cell identity. Long non-coding RNAs (lncRNAs) add further complexity by modulating miRNA networks through multiple mechanisms (Dinger et al , 2009; Guttman & Rinn, 2012; Statello et al , 2021). First, several lncRNAs serve as primary transcripts for miRNA biogenesis, directly producing small RNAs that influence stem cell fate. Second, lncRNAs can act as competing endogenous RNAs (ceRNAs), sequestering miRNAs away from their mRNA targets and thus sustaining pluripotency gene expression (Cesana et al , 2011; Tay et al , 2014). Several ceRNAs have been directly implicated in pluripotency control. For instance, the STAT3-regulated lncRNA Lncenc1 promotes self-renewal by sponging miR-128, thereby stabilizing Klf4 (Monteleone et al , 2024). In mESCs, the lncRNA Cyrano sustains pluripotency by titrating miR-7, thereby preventing repression of self-renewal pathways (Smith et al , 2017). In human ESCs, a regulatory feedback loop involves the Large Intergenic Non-Coding RNA – Regulator of Reprogramming ( LINC-ROR ), the microRNA miR-145, and the pluripotency transcription factors OCT4 , SOX2 , and NANOG : miR-145 inhibits OCT4 , SOX2 , and NANOG expression; in turn, these transcription factors promote LINC-ROR transcription; and the sponge domain of LINC-ROR titrates miR-145, preventing degradation of pluripotency transcripts (Loewer et al , 2010; Wang et al , 2013). Another example is the sponge lncRNA Growth Arrest Specific transcript 5 ( GAS5 ), which regulates NODAL signaling in hESCs. NODAL promotes self-renewal by activating OCT4 , SOX2 , and NANOG , and GAS5 expression is induced by OCT4 and SOX2 . In turn, GAS5 sequesters miR-2467-5p, miR-3200-3p, and miR-let7a/e-5p, all of which target NODAL, thereby reinforcing pluripotency (Xu et al , 2016). Together, these observations suggest that lncRNAs and miRNAs form intertwined circuits that buffer pluripotency against stochastic fluctuations while preserving responsiveness to differentiation cues. However, the repertoire of STAT3-regulated lncRNAs integrating both miRNA precursor and sponge activities remains poorly characterized. Here, we identify a novel STAT3 target lncRNA, Asgard ( Another Self-renewal GuARDian ), which is rapidly induced by LIF and enriched in the pluripotent epiblast. We show that Asgard is essential for efficient mESC self-renewal and operates through a dual mechanism: it acts as a precursor for the differentiation-promoting miRNA Odin , while simultaneously functioning as a sponge that sequesters Odin and related miRNAs. This duality positions Asgard as a unique integrative hub that connects STAT3 transcriptional inputs with post-transcriptional regulation of pluripotency. Results Identification of a novel LIF/STAT3 target gene as a potential regulator of pluripotency in mouse embryonic stem cells We used E14-S3ER cells to analyze the STAT3 transcriptome in mouse ESCs (Bourillot et al , 2009). These cells express a hormone-inducible mutant form of STAT3 (STAT3-ER T2 ), whose activity is specifically controlled by tamoxifen (Matsuda et al. , 1999). E14-S3ER cells can self-renew in presence of either LIF or 4’hydroxy-tamoxifen (4’OHT). Transcriptome analysis of E14-S3ER cells deprived of 4’OHT, followed by restimulation with either LIF or 4’OHT, identified 65 genes commonly regulated by both stimuli. Among them was an uncharacterized transcript corresponding to Affymetrix probe ID 1456160_at ( Fig. 1A ). RT-PCR confirmed that 1456160_at expression was activated by LIF or 4’OHT. A 1.5-fold upregulation upon 4’OHT stimulation in presence of cycloheximide indicated that 1456160_at is a direct STAT3 target gene in E14-S3ER cells ( Fig. 1A ). To validate this regulation in a different context, we examined 1456160_at expression in the E14tg2a mESC line after 24 h of LIF deprivation, followed by for 24 h of LIF restimulation. RT-PCR revealed a threefold reduction in expression after LIF withdrawal and a sevenfold induction upon LIF re-addition ( Fig. 1B ). We next assessed 1456160_at expression during embryoid body (EB) differentiation. Its transcript levels decreased as early as day 1, similar to the downregulation of key pluripotency factors Klf4 and Klf5 , but were not completely extinguished during differentiation ( Fig. 1C ). Download figure Open in new tab Figure 1. Identification of Asgard , a novel pluripotency regulator in mESCs. (A) Strategy for identifying STAT3 target genes. The Venn diagram shows genes induced by leukemia inhibitory factor (LIF), 4-hydroxytamoxifen (4’OHT), or both, including an uncharacterized gene corresponding to Affymetrix probe ID 1456160_at . Histogram shows 1456160_at expression in E14-S3ER cells treated with LIF or 4’OHT, measured by microarray and RT-PCR, normalized to untreated controls. (B) 1456160_at mRNA levels (ΔCt) in E14tg2a cells cultured with continuous LIF, deprived of LIF for 24 h, or restimulated with LIF for 2 h, normalized to untreated control. (C) Phase-contrast images of embryoid bodies (EBs) at day 4 of differentiation and RT-PCR analysis of 1456160_at , Klf4 , Klf5 , and β-actin expression during EB-mediated differentiation of E14tg2a cells. (D) Experimental workflow for testing 1456160_at function in mESC pluripotency. Histogram shows the proportion of undifferentiated (red), mixed (orange), and differentiated (beige) colonies in clonal assays after knockdown with two independent shRNAs ( pLenti6/BLOCK-iT-PGKneoR ). (E) qRT-PCR analysis of pluripotency and lineage markers ( Rex1 , Fgf5 , Sox17 , Gata4 , FoxA2 , Gata6 , Nodal , Brachyury , Mixl1 , Gsc ) in colonies from panel D. (F) Knockdown of 1456160_at ( Asgard ) in E14tg2a cells using shRNAs targeting regions distinct from the Affymetrix probe sequence. Histograms show colony number and area in clonal assays after Asgard knockdown. Data represent means ± SE from three independent experiments; Student’s t -test (* p < 0.05; ** p < 0.01; *** p < 0.001). To explore the functional role of 1456160_at in ESC self-renewal, we performed knockdown experiments using lentiviral vectors encoding two independent small hairpin (sh)RNA targeting 1456160_at ( sh1456160_at-1 and sh1456160_at-2 ), or a control shRNA targeting GFP, together with a neomycin resistant cassette. Twenty-four hours post-infection, E14tg2a ES cells were replated at clonal density and cultured for 7 days under G418 selection to eliminate non-infected cells. Knockdown efficiency was confirmed by RT-PCR ( Sup. Fig. 1 ). Colony-forming assays scored G418-resistant colonies as undifferentiated (alkaline phosphatase (AP) + , mixed (AP + /AP - ), or differentiated (AP - ). Control cells expressing shGFP maintained nearly 80% undifferentiated AP + colonies. In contrast, sh1456160_at-1 and sh1456160_at-2 reduced the proportion of AP + colonies by ∼ 45% and ∼ 30%, respectively, with a corresponding increase in mixed AP + /AP - colonies ( sh1456160_at-1, +5% ; sh1456160_at-2 , +30%) and AP - differentiated colonies ( sh1456160_at-1 , +40%, sh1456160_at-2 , +5%) ( Fig. 1D ). qRT-PCR analysis of pooled AP + , mixed, and AP - colonies revealed that 1456160_at knockdown reduced expression of the pluripotency marker Rex1 ( Zfp42 ), while inducing early ectoderm ( Fgf5 ) and endoderm markers ( Sox17 , FoxA2 , Gata4 and Gata6 ) ( Fig. 1E ). Collectively, these findings identify 1456160_at as a novel LIF/STAT3 target gene that promotes pluripotency and inhibits differentiation in mESCs. Based on its role, we renamed it Asgard (Another Self-renewal GuARDian). Download figure Open in new tab Supplementary figure 1: Validation of shRNA-mediated knockdown efficiency. qRT-PCR analysis of target gene expression following lentiviral transduction of mESCs with 1456160_at-shRNA1 , 1456160_at-shRNA2 , and Asgard-shRNA . Expression levels were normalized to those of cells transduced with a non-targeting control shRNA against GFP (shRNA-GFP). Asgard is a long non-coding RNA regulating mESC self-renewal The Affymetrix probe ID 1456160_at corresponds to a 407-nucleotide expressed sequence tag (EST) located on chromosome 11, in the antisense orientation within the first intron of the Nucleoredoxin ( Nxn ) gene ( Fig. 2A ). To determine the full-length sequence of the Asgard transcript, we performed RACE-PCR, obtaining a 766-nt fragment that was subsequently sequenced ( Sup. Fig. 2A ). Given its intronic location, we hypothesized that Asgard encodes a non-coding RNA. Northern blot analysis of RNA from wild-type E14tg2a mESCs–as well as from Dicer -/- and DGCR8 -/- mESCs defective in small RNA biogenesis (Kanellopoulou et al , 2005; Wang et al. , 2007)–was performed using a probe derived from the 766-nt fragment. Northern blotting revealed a ∼6,000-nt transcript corresponding to Asgard , with low expression in wild-type cells but markedly increased levels in Dicer⁻/⁻ and DGCR8⁻/⁻ mESCs ( Fig. 2B ). This increase was further supported by transcriptome analysis of Dicer⁻/⁻ mESCs ( Fig. 2E ). These data suggest that Asgard is a lncRNA subject to processing by Dicer and DGCR8. The complete Asgard sequence was reconstructed by sequencing RT-PCR products amplified with progressively extended 3′ and 5′ primers beyond the region defined by the initial RACE-PCR fragment ( Sup. Fig. 2B ). Sequence analysis using the NCBI ORFfinder tool ( https://www.ncbi.nlm.nih.gov/orffinder/ ) detected no long open reading frames, further supporting its classification as a lncRNA ( Sup. Fig. 4A ). Download figure Open in new tab Supplementary Figure 2: Experimental strategy for amplification of the full-length Asgard transcript. (A) Schematic representation of the RACE-PCR approach used to amplify a 766-nt fragment corresponding to the 1456160_at sequence. (B) RT-PCR-based strategy for obtaining the full-length Asgard transcript using 5′ and 3′ primers located outside the RACE-amplified region. (C) Principal component analysis (PCA) of transcriptomes from E14tg2a mESCs cultured in LIF/serum or 2i/LIF conditions, and Dicer -/- mESCs, using datasets from GSE263060, GSE215141, and GSE172282. (D) Principal component analysis (PCA) of transcriptomes from mouse morula (E2.5), inner cell mass (ICM; E3.5), and epiblast (E4.5) stages, using data from E-MTAB-2958. Download figure Open in new tab Figure 2. Expression pattern of Asgard in mESCs. (A) Sequence and genomic localization of Affymetrix probe ID 1456160_at within the Asgard locus, visualized using the UCSC Genome Browser ( https://genome-euro.ucsc.edu ). (B) Northern blot analysis of Asgard expression in wild-type (E14tg2a), DGCR8 -/- , and Dicer -/- mESCs. (C) In situ hybridization of E14tg2a cells cultured in LIF/serum or 2i/LIF using two independent RNAscope probes targeting Asgard . (D) qRT-PCR analysis of Asgard mRNA levels (ΔCt) in E14tg2a and CGR8 cells cultured in LIF/serum or 2i/LIF, normalized to LIF/serum. (E) Histogram representation of the mRNA level (DESeq2-normalized counts) of Asgard , in E14tg2a mESCs cultured in LIF/serum or 2I/LIF, and Dicer -/- mESCs. (F) Boxplot representation of the mRNA level (DESeq2-normalized counts) of Asgard , in mouse morula (E.2.5), mouse ICM (E3.5) and mouse epiblast (E4.5). (G) Fluorescent in situ hybridization (FISH) detection of Asgard in E4.5 mouse embryos using two distinct FISH probes. Statistical test: t-test. We next examined Asgard expression under different culture conditions. RNA in situ hybridization using two independent probes spanning the full transcript revealed higher expression in E14tg2a mESCs cultured in 2i/LIF compared to serum/LIF conditions ( Fig. 2C ). This enrichment was confirmed by qRT-PCR, showing an ∼8-fold increase in E14tg2a cells and a ∼4-fold increase in CGR8 cells under 2i/LIF ( Fig. 2D ), as well as by analysis of published RNA-seq datasets ( Fig. 2E , Sup. Fig. 2C ). Given that mESCs correspond to the preimplantation epiblast in vivo , we analyzed Asgard expression during early embryogenesis using published RNA-seq datasets. Expression was detected at the morula (E2.5), inner cell mass (E3.5), and epiblast (E4.5) stages, with a trend toward higher levels at earlier stages, although this difference did not reach statistical significance ( Fig. 2F , Sup. Fig. 2D ). Consistent with these data, fluorescent in situ hybridization (FISH) on E4.5 embryos using two independent probes revealed enrichment of Asgard in the epiblast compared to surrounding tissues ( Fig. 2G ), supporting its association with the naïve pluripotent state. To investigate Asgard function, we knocked down its expression using lentiviral shRNAs targeting sequences outside the original 1456160_at probe region ( pLenti6/BLOCK-iT-PGKneo-shAsgard ). Twenty-four hours post-infection, E14tg2a mESCs were replated at clonal density and cultured for 7 days under selection. Colonies were then fixed and stained for AP, and quantified. Asgard knockdown led to a marked reduction in both the number and size of AP-positive colonies ( Fig. 1F ), indicating a requirement for Asgard in maintaining mESC self-renewal. Asgard promoter is regulated by STAT3 in mouse embryonic stem cells To investigate the promoter activity of the Asgard gene, we examined ENCODE datasets ( https://www.encodeproject.org ). In the 5’ region of the gene, we identified a 630 bp sequence bearing characteristic promoter-associated epigenetic marks: strong RNA polymerase II occupancy, pronounced H3K4me3 enrichment, modest H3K27me3 signal, and no detectable H3K9me3 (Bernstein et al , 2006). This sequence, hereafter referred to as the “Sense promoter”, was cloned upstream of a luciferase reporter in the pGL4 ( pAsg-Sense-Luc ) ( Fig. 3A , Sup. Fig. 3A ). For comparison, a 3’ region displaying similar epigenetic features–termed the “Antisense promoter”–was cloned in the same configuration ( pAsg-Antisense-Luc ) ( Fig. 3A , Sup. Fig. 3A ). E14tg2a mESCs were transfected with pGL4-Luc (empty vector), pAsg-Sense-Luc , or pAsg-Antisense-Luc and cultured in three conditions: continuous LIF, 24 h LIF withdrawal, or LIF withdrawal followed by 2h LIF restimulation. Luciferase assays revealed that only the Sense promoter exhibited LIF-dependent activity, which decreased 1.7-fold upon LIF removal and increased 1.9-fold upon LIF restimulation ( Fig. 3B ). Similar results were obtained in E14-S3ER mESCs, which express a hormone-inducible STAT3-ER T2 variant that supports proliferation in the presence of 4’OHT. 4’OHT withdrawal and re-addition confirmed that the 630 bp Sense promoter mediates STAT3-dependent luciferase activation in E14-S3ER cells ( Fig. 3C ). Download figure Open in new tab Supplementary Figure 3: Functional analysis of STAT3 binding sites within the Asgard Sense promoter. (A) Nucleotide sequences of the “Sense” and “Antisense” promoter regions cloned into pGL4-Luc to generate the reporter constructs pAsg-Sense-Luc and pAsg-Antisense-Luc. (B) Sequences of the two putative STAT3 binding sites identified in the “Sense” promoter at positions 409 and 492, with mutations (in red) introduced into pAsg-Sense-Luc to generate the mutant constructs pAsg-Sense-ΔS3(409)-Luc and pAsg-Sense-ΔS3(492)-Luc . (C) Luciferase reporter assay in E14-S3ER mESCs transfected with either the wild-type or mutant constructs and cultured in the presence of 4’OHT. Data represent mean ± SE from three independent experiments. (D) Schematic representation of the pSB-TRE-CRISPRoff-EF1A-TetOn plasmid (Addgene #203355), enabling doxycycline-inducible expression of dCas9 fused to the DNMT3A and DNMT3L methyltransferases, as well as to the KRAB repression domain. Western blot analysis of dCas9 protein expression in the CrOff#1 clone of E14tg2a mESCs generated following transfection with the pSB-TRE-CRISPRoff-EF1A-TetOn plasmid and puromycin selection, cultured under –doxycycline and +doxycycline conditions. Phase-contrast images of CrOff#1 cells under –doxycycline and +doxycycline conditions. (E) Schematic representation of the pSB-BbsI-sgRNA plasmid (Addgene #203359), enabling expression of guide RNAs (sgRNAs). Table listing the sequences of five sgRNA spacer regions designed to target the Asgard gene promoter (designed using CRISPOR; https://crispor.gi.ucsc.edu ). These sequences were cloned into the pSB-BbsI-sgRNA plasmid. Statistical significance was determined by Student’s t -test (* p < 0.05; ** p < 0.01; *** p < 0.001). Download figure Open in new tab Figure 3. Characterisation of the Asgard promoter. (A) Chromatin landscape of the Asgard locus in Bruce4 mESCs. ChIP-seq binding profiles for RNA Polymerase II (Pol II), H3K4me3 (active promoter mark), and H3K9me3/H3K27me3 (repressive marks) were obtained from the ENCODE database and aligned to the Asgard genomic region. Two distinct sequences showing promoter-associated epigenetic features were identified: a 5′ “Sense Promoter” and a 3′ “Antisense Promoter”. Both sequences were cloned into the pGL4-Luc reporter plasmid. Schematic shows pGL4-Luc, pAsg-Sense-Luc, and pAsg-Antisense-Luc constructs. (B) Luciferase reporter assay in E14tg2a mESCs transfected with pGL4-Luc, pAsg-Sense-Luc , or pAsg-Antisense-Luc , under continuous LIF, 24 h LIF deprivation, or 2 h LIF re-stimulation. (C) Luciferase activity in E14-S3ER mESCs transfected with pGL4-Luc or pAsg-Sense-Luc under continuous 4’OHT, 24-h 4’OHT withdrawal, or 2 h 4’OHT re-stimulation. (D) In silico prediction of two STAT3 binding sites at positions 409 and 492 of the Sense Promoter ( JASPAR database). Schematic of mutant constructs pAsg-Sense-ΔS3(409)-Luc and pAsg-Sense-ΔS3(492)-Luc , with individual STAT3 site mutation. (E) Luciferase assay in E14tg2a mESCs transfected with wild-type ( pAsg-Sense-Luc ) or mutant ( pAsg-Sense-ΔS3(409)-Luc and pAsg-Sense-ΔS3(492)-Luc ) reporter plasmids, under LIF, 24 h LIF deprivation, or 2 h LIF re-stimulation. (F) ChIP-PCR analysis in E14tg2a mESCs using antibodies to total STAT3 or phosphorylated STAT3 (Y705). Primers targeted the Asgard promoter (S3) or a negative control region (Neg). Left: representative electrophoresis gel image; Right: quantification of PCR signal. (G) RT-PCR analysis of Asgard expression in CrOff #1-Gb and CrOff #1-Gc cells under –doxycycline and +doxycycline conditions. The positions of the PCR products along the Asgard gene are indicated in the schematic shown above. Data represent mean ± SE from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 ( Student’s t -test). JASPAR analysis ( https://jaspar.elixir.no ) identified two putative STAT3-binding sites within the Sense promoter at positions 409 and 492 ( Fig. 3D ). To assess their functional relevance in regards to the activation of the Asgard gene in response to LIF, each site was mutated by site-directed mutagenesis, generating pAsg-Sense-ΔS3(409)-Luc and pAsg-Sense-ΔS3(492)-Luc ) ( Fig. 3D , Sup. Fig. 3B ). After transfection into E14tg2a cells, luciferase assays revealed that mutation of the 492 site abolished LIF-induced promoter activation, whereas mutation of the 409 site enhanced the response to LIF by ∼ 2-fold ( Fig. 3E ). Similar results were obtained in E14-S3ER cells cultured with 4’OHT ( Sup. Fig. 3C ). To test whether STAT3 directly binds the Asgard promoter, we conducted ChIP-PCR in mESCs cultured with (+LIF) or without (-LIF, 24h) LIF. LIF stimulation induces phosphorylation of STAT3 on tyrosine 705, promoting its dimerization, nuclear translocation, and binding to the promoters of target genes (Zhong et al , 1994). Chromatin was immunoprecipitated using antibodies against total STAT3 or STAT3 phosphorylated on Tyr705 [P(Y705)-STAT3]. Primers flanking the STAT3-binding sites in Asgard promoter (S3 primers) and control primers targeting a STAT3-free region (neg primers) were used for PCR. In +LIF conditions, STAT3 binding was detected with both antibodies, with 2.5-fold (STAT3) and 5.5-fold [P(Y705)-STAT3] enrichment relative to no-antibody control ( Fig. 3F , lanes 1 and 3 vs. lane 5). No enrichment was observed with neg primers (lanes 2, and 4 vs. lane 6). LIF withdrawal reduced STAT3 binding by ∼ 4-fold (lane 7 vs. lane 1; lane 9 vs. lane 3), confirming LIF-dependent recruitment of STAT3 to the Asgard promoter region containing binding sites 1 and 2. To functionally validate the 630 bp Sense region as the Asgard promoter, we employed a CRISPR-based transcriptional repression approach (Gil et al., 2024). E14tg2a mESCs were engineered to express a doxycycline-inducible dCas9 fusion protein coupled to DNMT3A, DNMT3L, and the KRAB repressive domain ( Sup. Fig. 3D ), generating the CrOff #1 cell line. These cells were subsequently transfected with guide RNAs targeting the 630 bp Sense region ( Sup. Fig. 3E ) and subjected to hygromycin selection. Following doxycycline induction, Asgard expression was assessed by RT-PCR. Analysis of two independent clones, CrOff #1-Gb and CrOff #1-Gc, generated using guide RNAs b and c, respectively, revealed a consistent reduction in Asgard expression under +Dox conditions compared to −Dox controls ( Fig. 2G ). This decrease was observed with two primer pairs tested, which specifically amplify either the Odin miRNA or the sponge domain, and was more pronounced in the CrOff #1-Gc clone. Notably, both the sponge domain and Odin miRNA levels were concomitantly reduced. These results demonstrate that the 630 bp Sense region functions as the Asgard promoter and drives the coordinated expression of both the Odin miRNA and the sponge domain, indicating that they originate from a common transcriptional unit. Together, these results demonstrate that Asgard expression is regulated by LIF-dependent STAT3 binding to its Sense promoter, with the site at position 492 being essential for activation. Long non-coding RNA Asgard functions both as a microRNA precursor and a microRNA sponge The regulation of Asgard by Dicer and DGCR8 suggested that it may serve as a primary microRNA (pri-miRNA). Sequence analysis using the miRortho database ( http://cegg.unige.ch/mirortho/ ) revealed a region with strong homology to microRNA miR-1195 , with a highly significant e-value of 5.7 × 10⁻¹⁴ ( Fig. 4A ). Further analysis with the MirID algorithm ( http://datalab.njit.edu/RNAcenter/ ) classified this region as a putative pre-miRNA ( Sup. Fig. 4B ). Using the stem-loop RT-PCR protocol developed by Varkonyi-Gasic et al., we successfully detected the expression of the miRNA encoded by Asgard ( Fig. 4B ) (Varkonyi-Gasic et al , 2007). To assess its function, synthetic miRNA mimics were transfected into E14tg2a mESCs. qRT-PCR analysis showed downregulation of the pluripotency markers Nanog and Rex1 , along with upregulation of the early ectodermal marker Fgf5 and various mesodermal and endodermal markers ( Fig. 4C ). Overexpression of the miRNA via lentiviral transduction (OE-miRAsgard) and subsequent neomycin selection for 7 days yielded cells with a differentiated morphology. qRT-PCR confirmed reduced pluripotency marker expression and increased differentiation marker expression ( Fig. 4D ). These data indicate that the Asgard -encoded miRNA promotes differentiation. This miRNA was designated Odin ( One Differentiation INducer ). Download figure Open in new tab Supplementary Figure 4: In silico analysis of the coding potential and miRNA biogenesis capacity of the Asgard transcript. (A) Open reading frame (ORF) prediction using the NCBI ORFfinder tool ( https://www.ncbi.nlm.nih.gov/orffinder/ ). A total of 38 ORFs were identified, the longest spanning 360 nucleotides. (B) Computational prediction of the Asgard pre-miRNA sequence using the MirID algorithm ( http://datalab.njit.edu/RNAcenter/ ). The input sequence was classified as a putative pre-miRNA, supporting its potential role in miRNA biogenesis. Download figure Open in new tab Figure 4. The lncRNA Asgard functions as a miRNA sponge. (A) In silico prediction of a pre-miRNA hairpin and mature miRNA sequence within the Asgard gene (miROrtho database). (B) Detection of mature Asgard miRNA in E14tg2a mESCs cultured in LIF/FBS using stem-loop RT-PCR. (C) qRT-PCR analysis of pluripotency ( Rex1 , Nanog ) and differentiation ( Fgf5 , Brachyury , Mixl1 , Eomes , Gata6 , and PECAM1 ) markers in E14tg2a mESCs transfected with an Asgard miRNA mimic or control mimic. (D) Phase-contrast microscopy of E14tg2a mESCs overexpressing Asgard miRNA (OE-miRAsg) versus control cells. qRT-PCR analysis of pluripotency ( Nanog , Rex1 ) and differentiation ( Brachyury , Fgf5 , Gata4 , Gata6 , Dab2 , FoxA2 , Sox17 , Flk1 , Bmp2 , Hand1 , Msx2 ) markers in OE-miRAsg and control cells. (E) In silico prediction of miRNA response elements (MREs) for Odin within Asgard (Seagal database). (F) Identification of miRNAs homologous to the predicted Odin miRNA by aligning the Odin sequence against the mouse miRNA database (miRBase BLAST). Seed sequences are underlined in green. (G) Predicted binding of candidate miRNAs ( miR-7062-5p, miR-3470b, miR-378d, miR-6715-3p, miR-6363 , and miR-5118 ) to the Asgard sponge domain using RNA22v2 and miRNAsong. Free binding energies (kcal/mol) are indicated for each interaction. (H) Schematic of luciferase reporter construct pCAG-Luc-Sponge , in which the Asgard sponge domain was cloned into the 3′ UTR of luciferase under the CAG promoter. Luciferase activity was measured in E14tg2a mESCs co-transfected with pCAG-Luc-Sponge and either an Asgard miRNA mimic or a control mimic. The function of Odin as a differentiation inducer contrasts with the function of Asgard as a pluripotency stabilizer. To address this issue, we analyzed potential Odin targets using Segal lab tools ( http://132.77.150.113/pubs/mir07/mir07_prediction.html ), which revealed four putative Odin-binding sites clustered within a 500-nt region of the Asgard sequence ( Fig. 4E ). This suggested the existence of a “sponge domain” capable of sequestering Odin or related miRNAs. BLASTn searches against all annotated mouse miRNAs in miRBase release 22 (Kozomara et al , 2019) identified 10 miRNAs with sequence similarity to Odin : miR-1195 , miR-5118, miR-1198-3p, miR-3470a, miR-3470b, miR-7062-5p, miR-6363, miR-7651-5p, miR-378d , and miR-6715-3p ( Fig. 4E ). To predict binding sites for these Odin -related miRNAs within the Asgard sponge domain, we used two independent prediction tools–RNA22v2 ( https://cm.jefferson.edu/rna22/Interactive/ ) (Miranda et al , 2006) and miRNAsong ( https://www2.med.muni.cz/histology/miRNAsong/ ) (Barta et al , 2016)–and retained only sites predicted by both. Six miRNAs were predicted to bind the sponge domain: miR-7062-5p (four predicted binding sites), miR-3470b (two sites), and one site each for miR-378d, miR-6715-3p, miR-6363 , and miR-5118 ( Fig. 4G ). These predictions support the hypothesis that Asgard sequesters a subset of Odin -related miRNAs, particularly miR-7062-5p and miR-3470b . To functionally test this, the 500-nt sponge domain was cloned into the 3′ untranslated region (3′UTR) of the luciferase gene in pCAG-Luc , generating pCAG-Luc-Sponge . This construct was co-transfected into E14tg2a mESCs with either a negative control mimic or a synthetic Odin mimic. Luciferase assays revealed that the Odin mimic reduced reporter activity by 2-fold compared to the control ( Fig. 4H ), providing functional evidence that Asgard acts as a miRNA sponge, capable of sequestering Odin and related miRNAs to modulate their regulatory effects. Asgard sponge domain sustains mESC self-renewal We used the miRWalk interface ( http://mirwalk.umm.uni-heidelberg.de/search_mirnas/)(Sticht et al , 2018) to identify predicted target genes of miR-7062-5p, miR-3470b, miR-378d, miR-6715-3p, miR-6363 , and miR-5118 , followed by KEGG pathway enrichment analysis. Odin, together with these miRNAs, was predicted to target genes enriched in pathways essential for mESC pluripotency, including PI3K-AKT, JAK-STAT, MAPK, Wnt, p53, and canonical pluripotency networks ( Fig. 5A , Sup. Fig. 5A ). As most of these miRNAs are predicted to promote mESC differentiation, we hypothesized that Asgard may preserve self-renewal by sequestering them. To test this, we overexpressed the 500-nt sponge domain in mESCs using the pPB_Sponge plasmid. Two independent clones (E14 Sponge#1 and E14 Sponge#2 ) showed marked resistance to differentiation in LIF dilution and deprivation assays ( Fig. 5B , Sup. Fig. 5B ). Whereas control cells formed AP + colonies only at high LIF concentrations (> 100 U/mL), both E14 Sponge#1 and E14 Sponge#2 maintained AP + colonies at concentrations as low as 0.1 U/mL. Moreover, they retained AP + colony formation after 48 h of LIF-deprivation, while control cells lost this capacity after just 24 h. Furthermore, colony counts—a measure of clonogenic self-renewal—were significantly higher in sponge-overexpressing clones than in controls. At 1000 U/mL LIF, E14 Sponge#1 and E14 Sponge#2 generated 118 ± 5 and 104 ± 12 colonies, respectively, compared to 23 ± 6 in control E14tg2a cells ( Fig. 5C ). E14 Sponge#1 consistently exhibited the greatest clonogenic capacity, both in the presence and absence of LIF, and this enhancement persisted after prolonged LIF deprivation ( Fig. 5D ). Download figure Open in new tab Supplementary Figure 5: Functional pathway analysis of predicted miRNA targets and validation of Asgard sponge-expressing clones. (A) KEGG pathway enrichment analysis of predicted target genes for Odin , miR-3470b , miR-5118 , miR-6363 , and miR-378d , performed using the miRWalk platform. P -values were calculated using Fisher’s exact test to assess significant enrichment of KEGG pathways among the predicted targets. (B) PCR validation of six E14tg2a mESC clones generated by electroporation with the pPB-Sponge plasmid, followed by hygromycin selection and clonal expansion. Clones #1 and #2 were selected for further experiments and designated E14 Sponge#1 and E14 Sponge#2 , respectively. Download figure Open in new tab Figure 5. Overexpression of the Asgard sponge domain promotes mESC self-renewal under suboptimal culture conditions. (A) Predicted target signaling pathways of Odin, miR-3470b, miR-5118, miR-6363 , and miR-378d –miRNAs potentially regulated by the Asgard sponge domain and known to modulate mESC pluripotency. (B) Schematic of the experimental strategy for functional analysis of the Asgard sponge domain. The sponge sequence was cloned into a pPB-Hygromycin PiggyBac vector under the constitutive CAG promoter ( pPB-Sponge ) and stably integrated into E14tg2a mESCs. (C) Alkaline phosphatase (AP) staining of E14 Control cells and two independent Asgard sponge-expressing clones (E14 Sponge#1 and E14 Sponge#2 ) cultured at clonal density in media containing decreasing LIF concentrations. Right: quantification of AP + colonies and total colony number. (D) AP staining of E14 Control , E14 Sponge#1 , and E14 Sponge#2 cells cultured at clonal density without LIF for 0, 12, 24, 36, and 48 h, followed by re-stimulation with 1000 U/mL LIF for one week. Right: quantification of AP + colonies and total colony number in the clonal assay. Collectively, these findings indicate that the Asgard sponge domain enhances the robustness of pluripotency by buffering differentiation-promoting miRNAs. We propose a model in which a novel LIF target gene encodes a lncRNA that both generates and sequesters specific miRNAs, thereby fine-tuning the balance between self-renewal and differentiation ( Fig. 6 ). Download figure Open in new tab Figure 6. Proposed model for the role of the lncRNA Asgard in supporting mESC pluripotency. Schematic representation of the regulatory function of Asgard in maintaining pluripotency in mouse embryonic stem cell (mESCs). Asgard is transcriptionally activated by STAT3 signaling and contributes to pluripotency maintenance by acting as a molecular sponge for multiple microRNAs, including Odin and miR-3470b , which are predicted to target components of key pluripotency-associated signaling pathways. Through this dual mechanism—STAT3 dependent activation and miRNA sequestration— Asgard helps stabilize the pluripotent state. Discussion In this study, we identified Asgard , a previously uncharacterized lncRNA, as a direct transcriptional target of STAT3 and a novel regulator of pluripotency in mESCs. Asgard is rapidly induced upon LIF stimulation, and its depletion compromises self-renewal while promoting differentiation. Strikingly, Asgard integrates two antagonistic functions: it acts as a precursor for the differentiation-inducing microRNA Odin , while simultaneously functioning as a sponge that titrates Odin and related miRNAs away from their targets. This dual activity underscores a unique principle in stem cell regulation, where a single lncRNA transcript can both generate differentiation pressure and buffer it, thereby stabilizing the pluripotent state. Our findings extend the canonical view of STAT3 signaling in pluripotency. While STAT3 is well established as a master regulator of the transcriptional network that sustains self-renewal (Matsuda et al. , 1999; Niwa et al. , 2009), its role in controlling post-transcriptional layers of regulation has remained less clear. The discovery of Asgard highlights that STAT3 also reinforces pluripotency through non-coding RNA circuits, in which lncRNAs and miRNAs form intertwined feedback loops. Similar principles have been described for Lncenc1 , which sustains pluripotency by sponging miR-128 to stabilize Klf4 expression (Monteleone et al. , 2024). However, Asgard differs by embodying both miRNA precursor and lncRNA functions within the same transcript, positioning it as a self-contained regulatory module. The integration of miRNA and lncRNA functions within Asgard may provide an evolutionary advantage by ensuring robustness of pluripotency networks. On one side, the processing of Asgard into Odin miRNA provides a latent differentiation cue, priming the system for rapid lineage commitment when external signals reduce STAT3 activity. On the other, the sponge activity of Asgard buffers pluripotency factors from premature repression by Odin and related miRNAs. This architecture creates a fail-safe mechanism, in which pluripotency is maintained unless differentiation signals persistently outweigh the buffering capacity of the lncRNA. Such duality illustrates how lncRNAs can operate not merely as passive sponges or precursors, but as integrative hubs that coordinate multiple layers of RNA regulation. This dual mechanism also highlights broader implications for stem cell biology. miRNA–lncRNA crosstalk has been reported in diverse contexts (Statello et al. , 2021), but Asgard provides one of the first clear examples in ESCs where both functions converge in a single transcript. It raises the intriguing possibility that other STAT3-regulated lncRNAs may employ similar strategies, coupling miRNA production with miRNA sequestration to fine-tune pluripotency. Moreover, this mechanism could be conserved in other developmental systems, or even co-opted in pathological settings such as cancer, where aberrant STAT3 signaling and ncRNA regulation are common. Several questions remain open. The spectrum of miRNAs sequestered by Asgard requires further biochemical validation, as does the stoichiometric balance between its precursor and sponge functions. Whether these functions are temporally segregated—e.g., precursor activity during lineage priming and sponge activity during maintenance—also warrants investigation. In vivo , Asgard’s enrichment in the epiblast suggests a developmental role, but definitive proof will require genetic loss-of-function studies in embryos. Finally, it will be important to determine whether human pluripotent stem cells possess Asgard -like lncRNAs with dual regulatory activities. In conclusion, our study identifies Asgard as a dual-function lncRNA that integrates miRNA biogenesis and miRNA sequestration downstream of STAT3 signaling. By generating and simultaneously buffering differentiation-promoting miRNAs, Asgard provides a unique molecular strategy to stabilize the naïve pluripotent state while preserving developmental responsiveness. These findings reveal a new layer of complexity in non-coding RNA regulation and highlight how lncRNAs can act as integrative nodes that connect transcriptional and post-transcriptional control of cell fate. Materials and Methods Mouse ES cell culture and embryoid body formation Mouse embryonic stem cell (mESC) lines (E14, CGR8, Dicer -/- , and DGCR8 -/- ) were cultured at 37 °C in 7.5% CO₂ on 0.1% gelatin-coated plates. Cells were maintained in Glasgow’s Modified Eagle’s Medium (GMEM; Gibco, 21710025) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin–streptomycin (Gibco, 10378016), 2 mM glutamine, 1% non-essential amino acids (Gibco, 11140035), 1 mM sodium pyruvate (Gibco, 11360), 0.1 mM β-mercaptoethanol, and 1000 U/ml LIF. Cultures were passaged every 2 days by trypsinization and replated at a 1:5–1:10 ratio, with daily medium replacement. For serum-free culture, cells were maintained in N2B27 medium (StemCells, SCS-SF-NB-02) supplemented with 1 μM PD0325901 (Stemgent, 04-0004), 3 μM CHIR99021 (Stemgent, 04-0006), and 1000 U/ml LIF. Cells were seeded at high density (1.25 × 10⁶ cells per 9.5 cm² well) and passaged using Accutase (Life Technologies, A1110501). To induce differentiation, mESCs were cultured in suspension as hanging drops of LIF-free medium (∼100 cells per drop). After 3 days, embryoid bodies (EBs) were transferred to non-adherent Petri dishes on a rotary shaker and cultured for 7–10 days, with daily collection. RNA extraction and qPCR were performed as described below. For LIF titration assays, mESCs were plated at clonal density and cultured for 7 days in media containing decreasing concentrations of LIF (1000, 100, 10, 1, or 0.1 U/ml). For LIF withdrawal experiments, cells were seeded at clonal density and maintained without LIF for different time intervals (17, 24, 41, or 48 h), after which cultures were re-stimulated with 1000 U/ml LIF for 7 days. At the end of both assays, colonies were fixed, stained for alkaline phosphatase (AP) activity, and quantified. Plasmid construction shRNAs targeting Asgard were designed using the BLOCK-iT™ RNAi Designer (Thermo Fisher Scientific) and cloned into the pENTR™/U6 vector, followed by subcloning into pLenti6/BLOCK-iT-PGKpuroR . The resulting pLenti6-shRNA-puro plasmids were used to generate lentiviral particles for mESC transduction. Knockdown efficiency was assessed by quantitative real-time PCR, and the shRNA sequences with their silencing efficiencies are shown in Sup. Fig. 1 . The “Sense promoter” and “Antisense promoter” sequences ( Sup. Fig. 3A ) were PCR-amplified from E14tg2a mESC genomic DNA using primers containing XhoI restriction sites and inserted into the XhoI site of pGL4-Luc , generating pAsg-Sense-Luc and pAsg-Antisense-Luc . Mutations in the two STAT3 binding sites were introduced into pAsg-Sense-Luc using the QuikChange Site-Directed Mutagenesis Kit (Agilent, Cat. No. 200519), following the manufacturer’s instructions. The resulting mutant constructs were designated pAsg-Sense-ΔS3(409)-Luc and pAsg-Sense-ΔS3(492)-Luc . To generate pOE-miR-Asgard , the Odin sequence was cloned into pENTR™/U6 , followed by subcloning into pLenti6/BLOCK-iT-PGKpuroR . For pCAG-Luc , a CAG promoter fragment was PCR-amplified from pPB-HygroR using primers containing SacI and XhoI restriction sites, and subcloned into the corresponding sites of pGL4-Luc . The Asgard sponge domain sequence was PCR-amplified from E14tg2a mESC genomic DNA using primers with an XbaI site and inserted into pCAG-Luc , generating pCAG-Luc-Sponge . The Asgard sponge domain was also amplified from E14tg2a mESC genomic DNA with primers containing NheI and HpaI restriction sites and subcloned into the corresponding sites of pPB-HygroR , yielding pPB-Sponge . For CRISPRoff targeting, five independent sgRNA spacer sequences were designed to target the “Sense promoter” sequence using the CRISPick web portal ( https://crispor.gi.ucsc.edu ). Annealed sense and antisense oligonucleotides containing the appropriate BbsI overhangs were ligated into the BbsI site of the pSB-BbsI-sgRNA plasmid. Details of the spacer sequences are shown in Sup. Fig. 3E . RACE-PCR For 3′ RACE, 5 μg of total RNA was reverse-transcribed at 45 °C for 1 h with AMV reverse transcriptase using the GeneRacer® Oligo dT primer (Invitrogen). Two microliters of cDNA were amplified with the kit’s 3′ nested primer and a forward primer specific for EST 1456160_at using Platinum® Taq High Fidelity (Invitrogen). PCR conditions were: 94 °C for 2 min; 5 cycles of 94 °C for 30 s, 72 °C for 2 min; 5 cycles of 94 °C for 30 s, 70 °C for 2 min; 25 cycles of 94 °C for 30 s, 65 °C for 30 s, 68 °C for 2 min; final extension 68 °C for 10 min. Products were analyzed on 2% agarose gels. For 5′ RACE, 3 μg of RNA were dephosphorylated with CIP, decapped with TAP, and ligated to the GeneRacer™ RNA oligonucleotide. Reverse transcription was performed with AMV RT and random primers. PCR was carried out with the GeneRacer™ 5′ primer and an EST 1456160_at antisense primer using Platinum® Taq High Fidelity under touchdown conditions: 94 °C for 2 min; 5 cycles of 94 °C for 30 s, 72 °C for 4 min; 5 cycles of 94 °C for 30 s, 70 °C for 4 min; 25 cycles of 94 °C for 30 s, 65 °C for 30 s, 68 °C for 4 min; final extension 68 °C for 10 min. PCR products were cloned into pCR®4-TOPO® (Invitrogen), transformed into E. coli Top10, and selected on LB agar containing ampicillin. Colonies were expanded, plasmids purified with QIAprep Spin Miniprep (Qiagen), and verified by restriction digestion and sequencing. Lentiviral production and ES cell infection Lentiviral vectors expressing shRNAs or the Asgard microRNA were generated using the BLOCK-iT Lentiviral RNAi Expression System (Invitrogen). A total of 0.5 μg pLenti6/BLOCK-iT-PGKneor plasmid containing either shRNA or Asgard sequences was co-transfected with 1.5 μg ViraPower Packaging Mix (Invitrogen, K4975) and 6 μl Lipofectamine (Invitrogen, 11668) into 1 × 10⁶ 293T cells seeded in 2 ml medium. Viral supernatants were collected 48 h after transfection and clarified by centrifugation (3000 rpm, 10 min). E14 mESCs (1 × 10⁴ cells) were seeded in 24-well plates and infected with viral supernatants supplemented with 6 μg/ml polybrene and LIF. After 24 h, medium was replaced with fresh ES medium. At 48 h post-infection, cells were trypsinized, replated at 1 × 10³ cells per 10-cm dish, and cultured for 7 days in ES medium supplemented with 250 μg/ml G418 (Sigma, A1720). Resistant colonies were fixed with methanol and stained with Fast Red TR salt (Sigma) and naphthol solution in Tris-buffer (0.1 M, pH 9.2) to detect alkaline phosphatase activity. RNA extraction, RT-PCR and qPCR Total RNA was isolated using the RNeasy Mini Kit (Qiagen, 74106). Reverse transcription was performed with MuMLV-RT (Promega, M1701). For Asgard cloning, PCR was carried out using Phusion® High-Fidelity DNA Polymerase (NEB, M0530). qPCR was performed on cDNA using the LightCycler FastStart DNA Master SYBR Green kit (Roche, 12239264001), with expression levels normalized to β-actin. Transient transfection, luciferase assay and electroporation Transient plasmid transfections were performed using Lipofectamine® 2000 (Life Technologies, 11668019) in Opti-MEM (Life Technologies, 31985088), according to the manufacturer’s instructions. Cells were collected 24–48 h post-transfection for qPCR or luciferase assays. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Promega, E1910) on a GloMax® Multi Detection System (Promega), and firefly activity was normalized to Renilla to control for transfection efficiency. For electroporation experiments, 1.2 × 10⁶ E14tg2a mouse ES cells were co-electroporated with 3 μg of the pPB-Sponge-HygroR vector and 3 μg of a plasmid encoding the transposase, using the Neon® Transfection System 100 μl kit (Thermo Scientific, MPK10096). Electroporation was performed with two pulses of 20 ms at 1300 V. Cells were subsequently plated at low density in 100-mm dishes and cultured for one week in medium supplemented with 100 μg/ml hygromycin B to select for stable integrations. Sleeping Beauty (SB) transposon cell lines were generated by co-electroporating 1 × 10⁶ cells using the Neon Transfection System (Invitrogen; 1050 V, 20 ms, 2 pulses) with 2.5 μg of the SB transposon plasmid and 2.5 μg of the SB100X transposase-expressing vector pCMV(CAT)T7-SB100 (Addgene #127909). Stable transfectants were selected for 7 days using hygromycin (200 μg/ml) for the pSB-TRE-CRISPRoff-EF1A-TetOn construct (Addgene #203355) and puromycin (1 μg/ml) for the pSB-BbsI-sgRNA vector (Addgene #203359). Individual clones were isolated and screened for CRISPRoff dCas9 expression by western blot. Positive clones were maintained under hygromycin selection (200 μg/ml). CrOff #1-sgRNA clones were further validated by RT-PCR and subsequently cultured under dual selection with hygromycin (200 μg/ml) and puromycin (1 μg/ml). Northern blot analysis Total RNA (20 μg) was denatured at 65 °C and separated on 1.2% agarose gels containing 0.66% formaldehyde. RNAs were transferred overnight to Hybond-N+ membranes (Amersham) in 10× SSC, UV crosslinked, and hybridized at 68 °C with ^32P-labeled Asgard-specific probes. Membranes were washed sequentially with 1× SSC/0.1% SDS at room temperature and 0.5× SSC/0.1% SDS at 68 °C. Hybridization signals were detected by autoradiography. RNA In Situ Hybridization (RNAscope, Chromogenic Detection) Cells were cultured on poly-D-lysine coated coverslips and fixed in 4% paraformaldehyde for 30 min at room temperature. After PBS washes, cells were dehydrated in an ethanol series (50–100%) and stored at –20°C. Coverslips were rehydrated and permeabilized with RNAscope Protease III (ACD) for 15 min at room temperature. Target-specific RNAscope probes were hybridized using the RNAscope 2.5 HD Detection Kit (ACD) following the manufacturer’s instructions. Signal amplification was performed using the recommended amplification reagents, followed by chromogenic development with DAB substrate. Nuclei were counterstained with hematoxylin for 2 min, rinsed, and coverslips mounted with aqueous mounting medium. Images were acquired using bright-field microscopy and analyzed with ImageJ/Fiji. Fluorescent In Situ Hybridization (FISH) After flushing or dissection, embryos were fixed with 4% PFA in PBS overnight at 4°C and stored in methanol at −20°C. Embryos were rehydrated with 0.1% Tween 20 in PBS (PBT) and permeabilized with RIPA solution (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycolate, 0.1% SDS, 1 mM EDTA, 50 mM Tris [pH 8.0]) for 10–20 min at room temperature (RT). Embryos were postfixed with 4% PFA + 0.2% glutaraldehyde for 10 min at RT, and prehybridized in hybridization buffer (50% formamide, 5× SSC, 1% SDS, 100 mg/ml yeast tRNA, 50 mg/ml heparin) for 1 hr at 65°C. Hybridization with probes was done overnight at 65°C. Embryos were washed twice for 30 min with 50% formamide, 5× SSC, 1% SDS, once for 20 min with 50% formamide, 2× SSC, 1% SDS for 20 min at 65°C, and then transferred to PBT. Embryos were blocked with manufacturer’s blocking solution for 1–2 hr at RT. Embryos were treated with anti-DIG or -FITC antibody conjugated to peroxidase (1/200; Roche) overnight at 4°C. Fluorescent staining with TSA-Cy3, -Cy5 kits was carried out according to the manufacturer’s instructions (Perkin Elmer). Bioinformatics analysis All analyses were performed using R (version 4.5.3). Raw sequencing reads (FASTQ files) were pseudo-aligned to a modified mouse reference genome (GRCm38) supplemented with the Asgard sequence using the Salmon aligner ( https://github.com/COMBINE-lab/salmon ), run in quasi-mapping mode with sequence-specific bias correction and GC bias correction enabled. Transcript quantification was performed at default settings. Gene-level quantification and normalization were carried out with the DESeq2 package (version 1.48.2) (Love et al., 2014). Publicly available datasets used in this study include GSE215141, GSE172282, GSE263060, and E-MTAB-2958. Author contributions Investigation: V.G., F.P., Y.P., N.D., N.A., C.C., I.A., P-Y.B. Formal analysis: P-Y.B., P.S. Writing original draft, Funding acquisition: P-Y.B., P.S. Conceptualization, Supervision, Validation, Visualization, Project administration, Manuscript review and editing: P-Y.B., P.S. Acknowledgements This work was supported by the Fondation ARC pour la Recherche sur le Cancer (PJA-20151203436 to P-Y.B.), the Fondation pour la Recherche Médicale (EQU202303016295 to P.S.), the LabExs (ANR-10-LABX-73, REVIVE; ANR-10-LABX-0061, DEVweCAN; ANR-11-LABX-0042, CORTEX), and the University of Lyon within the program “Investissements d’Avenir” (ANR-11-IDEX-0007). Funder Information Declared Fondation ARC pour la Recherche sur le Cancer , PJA-20151203436 Fondation pour la Recherche Médicale , EQU202303016295 LabEx REVIVE , ANR-10-LABX-73 LabEx DEVweCAN , ANR-10-LABX-0061 LabEx CORTEX , ANR-11-LABX-0042 Investissements d’Avenir : University of Lyon , ANR-11-IDEX-0007 Footnotes $ Co-first authors This version has been revised to update fig.2, fig.3, sup. fig.2 and sup. fig.3, as well as the corresponding sections of the manuscript. Litterature 1. Aksoy I , Giudice V , Delahaye E , Wianny F , Aubry M , Mure M , Chen J , Jauch R , Bogu GK , Nolden T et al. ( 2014 ) Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells . Nat Commun 5 : 3719 OpenUrl CrossRef PubMed 2. Barta T , Peskova L , Hampl A ( 2016 ) miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico . Sci Rep 6 : 36625 OpenUrl PubMed 3. Bernstein BE , Mikkelsen TS , Xie X , Kamal M , Huebert DJ , Cuff J , Fry B , Meissner A , Wernig M , Plath K et al. ( 2006 ) A bivalent chromatin structure marks key developmental genes in embryonic stem cells . Cell 125 : 315 – 326 OpenUrl CrossRef PubMed Web of Science 4. Bourillot PY , Aksoy I , Schreiber V , Wianny F , Schulz H , Hummel O , Hubner N , Savatier P ( 2009 ) Novel STAT3 Target Genes Exert Distinct Roles in the Inhibition of Mesoderm and Endoderm Differentiation in Cooperation with Nanog . Stem Cells 27 : 1760 – 1771 OpenUrl CrossRef PubMed Web of Science 5. Cesana M , Cacchiarelli D , Legnini I , Santini T , Sthandier O , Chinappi M , Tramontano A , Bozzoni I ( 2011 ) A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA . Cell 147 : 358 – 369 OpenUrl CrossRef PubMed Web of Science 6. Dinger ME , Amaral PP , Mercer TR , Mattick JS ( 2009 ) Pervasive transcription of the eukaryotic genome: functional indices and conceptual implications . Brief Funct Genomic Proteomic 8 : 407 – 423 OpenUrl CrossRef PubMed 7. Guttman M , Rinn JL ( 2012 ) Modular regulatory principles of large non-coding RNAs . Nature 482 : 339 – 346 OpenUrl CrossRef PubMed Web of Science 8. Judson RL , Babiarz JE , Venere M , Blelloch R ( 2009 ) Embryonic stem cell-specific microRNAs promote induced pluripotency . Nat Biotechnol 27 : 459 – 461 OpenUrl CrossRef PubMed Web of Science 9. Kanellopoulou C , Muljo SA , Kung AL , Ganesan S , Drapkin R , Jenuwein T , Livingston DM , Rajewsky K ( 2005 ) Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing . Genes Dev 19 : 489 – 501 OpenUrl Abstract / FREE Full Text 10. Kozomara A , Birgaoanu M , Griffiths-Jones S ( 2019 ) miRBase: from microRNA sequences to function . Nucleic Acids Res 47 : D155 – D162 OpenUrl CrossRef PubMed 11. Loewer S , Cabili MN , Guttman M , Loh YH , Thomas K , Park IH , Garber M , Curran M , Onder T , Agarwal S et al. ( 2010 ) Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells . Nat Genet 42 : 1113 – 1117 OpenUrl CrossRef PubMed Web of Science 12. Martello G , Bertone P , Smith A ( 2013 ) Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor . EMBO J 32 : 2561 – 2574 OpenUrl Abstract / FREE Full Text 13. Matsuda T , Nakamura T , Nakao K , Arai T , Katsuki M , Heike T , Yokota T ( 1999 ) STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells . Embo J 18 : 4261 – 4269 . OpenUrl Abstract / FREE Full Text 14. Melton C , Judson RL , Blelloch R ( 2010 ) Opposing microRNA families regulate self-renewal in mouse embryonic stem cells . Nature 15. Miranda KC , Huynh T , Tay Y , Ang YS , Tam WL , Thomson AM , Lim B , Rigoutsos I ( 2006 ) A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes . Cell 126 : 1203 – 1217 OpenUrl CrossRef PubMed Web of Science 16. Monteleone E , Corrieri P , Provero P , Viavattene D , Pulvirenti L , Raggi L , Carbognin E , Bianchi ME , Martello G , Oliviero S et al. ( 2024 ) STAT3-dependent long non-coding RNA Lncenc1 contributes to mouse ES cells pluripotency via stabilizing Klf4 mRNA . Brief Funct Genomics 23 : 651 – 662 OpenUrl PubMed 17. Nichols J , Smith A ( 2009 ) Naive and primed pluripotent states . Cell Stem Cell 4 : 487 – 492 OpenUrl CrossRef PubMed Web of Science 18. Niwa H , Ogawa K , Shimosato D , Adachi K ( 2009 ) A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells . Nature 460 : 118 – 122 OpenUrl CrossRef PubMed Web of Science 19. Smith KN , Starmer J , Miller SC , Sethupathy P , Magnuson T ( 2017 ) Long Noncoding RNA Moderates MicroRNA Activity to Maintain Self-Renewal in Embryonic Stem Cells . Stem Cell Reports 9 : 108 – 121 OpenUrl PubMed 20. Statello L , Guo CJ , Chen LL , Huarte M ( 2021 ) Gene regulation by long non-coding RNAs and its biological functions . Nat Rev Mol Cell Biol 22 : 96 – 118 OpenUrl CrossRef PubMed 21. Sticht C , De La Torre C , Parveen A , Gretz N ( 2018 ) miRWalk: An online resource for prediction of microRNA binding sites . PLoS One 13 : e0206239 OpenUrl CrossRef PubMed 22. Tay Y , Rinn J , Pandolfi PP ( 2014 ) The multilayered complexity of ceRNA crosstalk and competition . Nature 505 : 344 – 352 OpenUrl CrossRef PubMed Web of Science 23. van Oosten AL , Costa Y , Smith A , Silva JC ( 2012 ) JAK/STAT3 signalling is sufficient and dominant over antagonistic cues for the establishment of naive pluripotency . Nat Commun 3 : 817 OpenUrl CrossRef PubMed 24. Varkonyi-Gasic E , Wu R , Wood M , Walton EF , Hellens RP ( 2007 ) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs . Plant Methods 3 : 12 OpenUrl CrossRef PubMed 25. Wang Y , Medvid R , Melton C , Jaenisch R , Blelloch R ( 2007 ) DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal . Nat Genet 39 : 380 – 385 OpenUrl CrossRef PubMed Web of Science 26. Wang Y , Xu Z , Jiang J , Xu C , Kang J , Xiao L , Wu M , Xiong J , Guo X , Liu H ( 2013 ) Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal . Dev Cell 25 : 69 – 80 OpenUrl CrossRef PubMed Web of Science 27. Xu C , Zhang Y , Wang Q , Xu Z , Jiang J , Gao Y , Gao M , Kang J , Wu M , Xiong J et al. ( 2016 ) Long non-coding RNA GAS5 controls human embryonic stem cell self-renewal by maintaining NODAL signalling . Nat Commun 7 : 13287 OpenUrl CrossRef PubMed 28. Zhong Z , Wen Z , Darnell JE , Jr . . ( 1994 ) Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6 . Science 264 : 95 – 98 OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted April 30, 2026. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency Vincent Giudice , Florence Perold , Yannicke Pijoff , Nathalie Doerflinger , Nicolas Allègre , Claire Chazaud , Irène Aksoy , Pierre Savatier , Pierre-Yves Bourillot bioRxiv 2025.09.23.677977; doi: https://doi.org/10.1101/2025.09.23.677977 Share This Article: Copy Citation Tools A STAT3-regulated lncRNA integrates microRNA biogenesis and sequestration to safeguard naïve pluripotency Vincent Giudice , Florence Perold , Yannicke Pijoff , Nathalie Doerflinger , Nicolas Allègre , Claire Chazaud , Irène Aksoy , Pierre Savatier , Pierre-Yves Bourillot bioRxiv 2025.09.23.677977; doi: https://doi.org/10.1101/2025.09.23.677977 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)