Dysregulation of U12-Type Splicing in Lupus Neutrophils

Dysregulation of U12-Type Splicing in Lupus Neutrophils | 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 Dysregulation of U12-Type Splicing in Lupus Neutrophils Luz P. Blanco , Binod Regmi , Carmelo Carmona-Rivera , Yudong Liu , Xiantao Wang , Philip M. Carlucci , Monica M. Jackson , Zerai Manna , Sarfaraz Hasni , Markus Hafner , Hong-Wei Sun , View ORCID Profile Mariana J. Kaplan doi: https://doi.org/10.1101/2025.11.06.686965 Luz P. Blanco 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Binod Regmi 2 Biodata Mining and Discovery Section, Office of Science and Technology, National Institutes of Health (NIH) , Bethesda, Maryland, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carmelo Carmona-Rivera 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yudong Liu 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA MD, PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiantao Wang 3 RNA Molecular Biology Laboratory , Diseases, National Institutes of Health (NIH) , Bethesda, Maryland, USA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Philip M. Carlucci 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Monica M. Jackson 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zerai Manna 4 Lupus Clinical Trials Unit, at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH) , Bethesda, Maryland, USA MSc. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sarfaraz Hasni 4 Lupus Clinical Trials Unit, at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH) , Bethesda, Maryland, USA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Markus Hafner 3 RNA Molecular Biology Laboratory , Diseases, National Institutes of Health (NIH) , Bethesda, Maryland, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hong-Wei Sun 2 Biodata Mining and Discovery Section, Office of Science and Technology, National Institutes of Health (NIH) , Bethesda, Maryland, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: sunh1{at}nih.gov mariana.kaplan{at}nih.gov Mariana J. Kaplan 1 Systemic Autoimmunity Branch, National Institutes of Health (NIH) , Bethesda, Maryland, USA 4 Lupus Clinical Trials Unit, at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH) , Bethesda, Maryland, USA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mariana J. Kaplan For correspondence: sunh1{at}nih.gov mariana.kaplan{at}nih.gov Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Objective Neutrophil dysfunction is a hallmark of systemic lupus erythematosus (SLE), but its molecular basis remains unclear. This study explores transcriptional and post-transcriptional changes in low-density granulocytes (LDGs), a proinflammatory neutrophil subset expanded in SLE, focusing on NADPH oxidase (Nox) function and minor intron splicing. Methods LDGs and normal-density neutrophils (NDGs) were isolated from SLE patients and healthy controls (HCs). CYBA (P22phox) expression was evaluated at transcript and protein levels. Nox activity was measured using luminol assays. Bulk RNA sequencing and rMATS software were used to assess alternative splicing, particularly of U12-type intron-containing genes. Results CYBA expression was reduced in SLE LDGs (n=11) compared to SLE NDGs and HCs (n=6), with levels resembling those in chronic granulomatous disease neutrophils. SLE LDGs exhibited impaired Nox activity (n=7 SLE, n=12 HC). CYBA is a U12 intron-containing gene, and transcriptomic analysis revealed broad downregulation of this gene class in SLE LDGs, suggesting minor spliceosome dysfunction. rMATS analysis showed increased U12-type intron retention and widespread splicing defects— including exon skipping and mutually exclusive exon use—in genes such as GBP5, MAEA and STX10 . These abnormalities were validated in an independent long-read RNA-seq dataset from SLE PBMCs. Importantly, splicing disruptions correlated with disease activity and autoantibody profiles. Conclusion Impaired U12-dependent splicing may contribute to neutrophil dysfunction in SLE, potentially via defective oxidative burst and altered immune regulation. These findings highlight the minor spliceosome as a novel player in lupus pathogenesis. Introduction The pathogenesis of systemic lupus erythematosus (SLE) remains incompletely understood, particularly regarding the role of aberrant RNA splicing in disease progression ( 1 ). Widespread splicing abnormalities have been reported in SLE, though it remains unclear whether these disruptions directly generate autoantigens or arise secondarily from chronic autoimmunity, including the production of autoantibodies against nucleic acids and spliceosomal proteins. Notably, autoantibodies targeting spliceosome proteins have been identified in multiple autoimmune conditions, including SLE and murine models of lupus ( 2 – 9 ). Splicing is a complex regulatory process required for the generation of mature mRNA. While most human genes are processed by the major U2 spliceosome, a small subset (∼700 genes) require the minor U12 spliceosome machinery. These U12-dependent genes are evolutionarily conserved, show tissue-specific expression, and are essential for key biological functions including stress response, immune regulation, and myeloid cell differentiation ( 10 – 24 ). Dysregulated alternative splicing in SLE affects a broad range of immune genes, including CD44, CD3 ζ , CTLA4, CREM, Fas , and DUSP22 , among others, thereby altering signaling, apoptosis, and tolerance pathways( 25 – 30 ) Neutrophils are central to SLE pathogenesis through the production of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs). A distinct subset of low-density granulocytes (LDGs) is expanded in SLE and displays enhanced NET formation, mitochondrial ROS production and distinct pathogenic phenotype ( 31 – 33 ). However, the molecular mechanisms driving their altered function remain incompletely defined. The CYBA gene encoding p22 phox , is essential for the stability and activity of multiple NADPH (Nox) complexes( 34 , 35 ). Reduced CYBA expression destabilizes Nox enzymes and impairs oxidative burst, linking defective redox signaling to immune dysregulation( 36 ). In this study, we identify impaired Nox activity in SLE LDGs, accompanied by reduced CYBA expression and evidence of U12 intron retention. Transcriptomic analysis further revealed broad splicing abnormalities in SLE LDGs, particularly affecting U12-type introns. These findings suggest that defective minor spliceosome activity may underlie neutrophil dysfunction and contribute to systemic autoimmunity. Materials and methods Neutrophils and LDG isolation Neutrophils and LDGs were isolated from peripheral venous blood of SLE subjects and healthy controls (HC), as described ( 33 , 37 ). SLE patients met revised American College of Rheumatology criteria for this disease ( 38 ) and were enrolled in protocol NIH#94-AR-0066. Healthy control volunteers were recruited at the Blood Bank Clinical Center, NIH. All subjects signed informed consent. Western blot, immunofluorescence, and flow cytometry Western blot was performed as previously described ( 39 ). Briefly, total protein (50 ug) was resolved in a 4–12% NuPAGE Bis-Tris gradient gel (Invitrogen). Proteins were immobilized onto a nitrocellulose membrane (Invitrogen) and blocked with 10% bovine serum albumin for 30 minutes at room temperature. The membrane was incubated with rabbit anti-human P22phox rabbit polyclonal (1:500; sc-20781, Santa Cruz Biotechnology) or rabbit polyclonal antibody against MPO (1:1,000; A0398, DAKO) overnight at 4°C. After 3 washes with PBS–Tween, the membrane was probed with secondary IRDye800CW goat anti-rabbit antibody (1:10,000; 926-32211, Li-Cor Biosciences) for 1 hour at room temperature. Proteins were detected using a Li-Cor Biosciences Odyssey Infrared Imaging System, in accordance with the manufacturer’s instructions. For immunofluorescence, neutrophils were seeded into a coverslip and incubated with P22phox rabbit polyclonal (1:500; sc-20781 Santa Cruz Biotechnology) for 1h at room temperature, washed briefly with PBS and incubated with a secondary anti rabbit Alexa 428 antibody (1:250; A21206, Invitrogen) for 1 h at room temperature. After a brief wash in PBS, the cells were fixed in 4% paraformaldehyde overnight at 4°C. After washing in PBS, the coverslips were mounted for fluorescent microscopy analysis using ProLong Gold (P36930, Invitrogen) and visualized using in a Leica DMI4000 B inverted microscope. Images were acquired using the microscope’s associated software. For the flow cytometry analysis, neutrophils or PBMCs were stained in FACS buffer (PBS and 2% fetal bovine serum) incubated with 100 μL human TruStain FcX on ice (1:50 dilution) for 10 min. After Fc blocking, the cells were incubated with anti-CD15 antibodies (Biolegend), followed by permeabilization with the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). The cells were then stained with anti-P22phox antibody or isotype control antibody (Santa Cruz Biotechnology), followed by staining with fluorescent-conjugated secondary antibodies. Cells were acquired on a BD LSR-Fortessa or BD FACSCanto. Data were analyzed using FlowJo software. Nox activity Nox activity of NDGs and LDGs was measured detecting chemiluminescence produced by luminol once oxidized by Nox in a plate assay. The activity was induced using either N-formyl-Met-Leu-Phe (fMLF) (4 x 10^6 M) or phorbol 12-myristate 13-acetate (PMA) (400 ng/ml). Luminol protected from light was used at 400 uM. A total of 100,000 cells in 100 ul/ well were aliquoted in HBSS containing 10 mM HEPES pH 7.3-7.4. Cells were preincubated with luminol (50 ul) for 10 min at 37°C and then with the activators (50 ul), and luminescence was measured over time up to 30 min and every minute in a Bi-Tek plate reader. The blank control was media alone and cells without activators. The activity is reported as luminescence units (RLU). Total RNA sequence RNAseq libraries were prepared from 1 µg of total RNA using the NEBnext Ultra Directional RNA Library kit (E7760) after ribosomal RNA depletion with the NEB Next rRNA Depletion kit (E7405). RNA-seq data were generated using Illumina’s HiSeq 3000 system. Raw sequencing data were processed with bcl2fastq v2.17.1 to generate FastQ files. Adapter sequences were removed using Trimgalore v0.4.2. Single-end reads of 50 bases were mapped to the human transcriptome and genome hg19 using TopHat v2.1.1. Gene expression values (RPKM: Reads Per Kilobase exon per Million mapped reads) were calculated using Partek Genomics Suite 6.6, which was also used for principal component analysis (PCA) and analysis of variance (ANOVA). Bam files generated by TopHat were used for downstream alternative RNA splicing analysis. Alternative Splicing Analysis Alternative splicing events were analyzed using rMATS v4.3 ( 40 ). Multiple unpaired comparisons were performed, with three to ten replicates in the first group (HC) and two to ten replicates in the second group (SLE-LDG, SLE-NDG, SLE-PBMC). rMATS identifies event-based alternative splicing across five patterns: skipped exon (SE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE), and retained intron (RI). Significant events were defined by FDR < 0.2( 41 ). These significant alternative splicing events were categorized into two groups based on whether the associated genes were part of the common spliceosome (U2-type) or the rare spliceosome (U12-type). A proportion test was performed to calculate the estimated z-score and p-value, assessing the relative frequency of events in U2- and U12-type genes compared to the total number of genes annotated as U2- or U12-type. Pathway analysis Statistically significant genes with splicing events were analyzed to obtain the relevant pathway plots using the Enrichr web site ( https://maayanlab.cloud/Enrichr/ ) and Appyter viewer ( 42 – 44 ). Statistical analysis The data in figures 2 and 4 was analyzed and plotted using GraphPad Prism software. Results To investigate neutrophil abnormalities in SLE, we first analyzed the expression of CYBA (P22phox), a component of the Nox complex. Western blot analysis revealed reduced CYBA protein levels in lupus LDGs compared to autologous NDGs or heathy control NDGs. Notably, CYBA expression in SLE LDGs was comparable to levels observed in chronic granulomatous disease (CGD) patients ( Figure 1A ), a condition associated with severely impaired Nox activity ( 45 ). Correspondingly, luminol-based assays demonstrated significantly impaired Nox activity in SLE LDGs, with reduced response to both fMLF and PMA stimulation, in contrast to autologous NDGs or healthy control NDGs ( Figure 1B ). Reduced CYBA (P22phox) expression in SLE LDGs was corroborated by both immunofluorescence staining ( Figure 1C ) and flow cytometry analysis ( Figure 1D ). Download figure Open in new tab Figure 1: Reduced expression of Nox component CYBA (P22phox) in SLE neutrophils A) Western blot (upper and middle and respective densitometry analysis (lower) showing the expression of CYBA (P22phox) and myeloperoxidase (MPO, as a house keeping protein) in neutrophils from healthy controls (n=6), SLE (n=11) or CGD (n=2) patients. The top Western gel compares LDGs with autologous NDGs whereas the bottom Western represents NDGs only; * p<0.05. B) Nox activity measured after stimulation with either fMLF (upper graph) or PMA (lower graph) in neutrophils obtained from SLE patients (n=7) or healthy controls (n=12). Asterisks in fMLF indicate statistically significant difference between lupus LDG and NDG, and in PMA significant difference between LDG and healthy control NDG; (*p<0.05), applied Mann-Whitney U-test. C) Images of neutrophils stained with an antibody against P22phox and a secondary antibody (FITC conjugated, green). Nuclei are stained blue with Hoechst (magnification 40x). D) Expression of P22phox in neutrophils measured by flow cytometry. Results represent mean + SEM. Of SLE patients whose samples were tested for CYBA expression and activity, 83% were anti-ANA and/or anti-ENA positive (50% anti-Ro, 33% anti-Sm, 66% anti-RNP, none anti-La) and mean SLEDAI was 3.6 + 3. To explore mechanisms underlying CYBA dysregulation, we analyzed total RNA sequencing data. CYBA mRNA expression was lower in SLE LDGs than in NDGs from SLE patients or controls ( Figure 2A ). Intron retention of CYBA , and U12-type minor intron number 2, showed increased retention in SLE LDGs, which inversely correlated with CYBA transcript levels ( Figure 2B ). This was not observed in the housekeeping gene ACTB , which showed minimal intron retention and no significant expression difference across cell types ( Figure 2C–D ). Similarly, the U12-containing gene PTEN exhibited reduced expression and increased intron retention in SLE LDGs ( Figure 2E–F ). Overall, genes harboring U12-type introns were significantly downregulated in SLE LDGs ( Figure 2G ), consistent with a global impairment of minor spliceosome function. Download figure Open in new tab Figure 2: SLE neutrophils display intron retention in genes containing a U12 intron A) Expression of CYBA mRNA in SLE or healthy control neutrophils. B) Intron retention in CYBA gene measured in SLE and healthy control neutrophils. C) Intron retention in the ACTB housekeeping gene (all U2) and the respective mRNA expression in D). E) PTEN mRNA expression and respective intron retention depicted in F). U12 containing genes expression in SLE and healthy control neutrophils in G). Results represent mean + SEM. *: p <0.05, ****: p <0.0001. Data analyzed using the Mann-Whitney U-test. Of SLE patients whose samples were sequenced, 86% were ANA positive, 43% were anti-dsDNA positive, 71% were anti-ENA positive (43% anti-Sm positive, 57% anti-Sm/RNP positive, 43% anti-Ro positive and 29% anti-La positive) and mean SLEDAI was 4 + 3.5. To test this hypothesis, we used rMATS to analyze alternative splicing events in RNA-seq data from SLE and control neutrophils. Consistent with prior reports, neutrophils from SLE patients (both LDGs and NDGs) exhibited an increased frequency of splicing anomalies. Notably, these disruptions were significantly enriched in genes containing U12-type introns compared to those with U2-type introns ( Table 1 ). Similar splicing patterns were observed in a publicly available long-read RNA-seq dataset of peripheral blood mononuclear cells (PBMCs) from SLE patients ( 46 ) suggesting that U12-type splicing dysregulation in SLE may be a systemic feature extending beyond the neutrophil compartment. View this table: View inline View popup Download powerpoint Table 1: Splicing events are significantly enhanced in U12 genes and in SLE derived cells rMATS analysis identified five categories of aberrant splicing: skipped exons (SE), mutually exclusive exons (MXE), intron retention (RI), and alternative 3’ and 5’ splice sites (A3SS, A5SS). Genes with significant splicing alterations are listed in Table 2 and Supplementary Data (S1–S3). Pathway analysis of these genes (Bioplanet 2019, KEGG 2021) revealed enrichment in immune-related pathways, including interferon and interleukin signaling ( Figure 3A ), chemokine signaling and MAPK/MEK pathways ( Figure 3B ), and pathways related to splicing regulation ( Figure 3C ). Download figure Open in new tab Figure 3. Pathway analysis of significantly regulated genes in SLE neutrophils Enrichment analysis of differentially expressed or spliced genes was performed using Bioplanet 2019 and KEGG 2021 (Human) pathway databases via the Enrichr platform ( https://maayanlab.cloud/Enrichr/ ) and Appyter viewer. Results are shown for the following comparisons: (A) Healthy control NDGs vs. SLE LDGs; (B) SLE LDGs vs. SLE NDGs, (C) Healthy control NDGs vs. SLE NDGs ( Table 2 for full list). View this table: View inline View popup Download powerpoint Table 2: Significant (FDR<0.2) U12 intron containing genes with their respective splicing event Beyond CYBA, we identified splicing disruptions in other components of the Nox complex ( CYBB , NCF1, NCF2 , and NCF4 ) across datasets (Supplementary Data S1– S4). Western blot and flow cytometry analyses confirmed reduced, although not statistically significant, levels of CYBB (P47phox) protein in SLE LDGs compared to SLE NDGs and healthy controls (Supplementary Figure S1). Finally, we investigated the clinical relevance of aberrant splicing events in SLE neutrophils. Splicing anomalies in LDGs showed the strongest associations with clinical disease activity, as measured by SLEDAI score, and with key autoantibody profiles, including anti-dsDNA and anti-Sm ( Figure 4A ; Supplementary Data S5). Splicing alterations in NDGs were more closely associated with the presence of total anti-ENA and of anti-SmRNP antibodies but did not correlate with disease activity scores ( Figure 4B ). These findings indicate that splicing dysregulation in LDGs may play a particularly important functional and clinical role in SLE. Download figure Open in new tab Figure 4. Correlation between clinical and laboratory parameters and splicing events in SLE neutrophils Pearson correlation coefficients between clinical and laboratory measures and splicing event types in: (A) SLE LDGs vs. healthy control (HC) NDGs and (B) SLE NDGs vs. HC NDGs. Splicing event types include: A3SS: alternative 3′ splice site; A5SS: alternative 5′ splice site; RI: intron retention; SE: exon skipping; MXE: mutually exclusive exon usage. Parameters analyzed: C3: complement component 3; C4: complement component 4; SLEDAI: SLE Disease Activity Index; ANA: antinuclear antibodies; Anti-ENA: anti-extractable nuclear antigens; Anti-SmRNP: anti-Smith ribonucleoprotein antibodies; SS-A/Ro and SS-B/La: autoantibodies targeting Ro and La proteins, respectively; Anti-CCP: anti-cyclic citrullinated peptide antibodies. Taken together, our results demonstrate widespread alternative splicing abnormalities in SLE neutrophils, with a specific enrichment of defects in U12-type introns. The pronounced splicing disruptions observed in LDGs, especially in genes relevant to immune regulation and oxidative function, may contribute to their pathogenic role and implicate minor spliceosome dysfunction as a mechanistic contributor to SLE pathogenesis. Discussion Our study reveals that neutrophils from SLE patients, especially LDGs, exhibit pervasive splicing abnormalities, with a striking enrichment of aberrant splicing in genes containing U12-type minor introns. These splicing defects likely have profound functional consequences, contributing to altered neutrophil phenotypes and promoting SLE pathogenesis. Disruptions in U12 intron splicing may impair neutrophil function through multiple mechanisms. First, defective splicing could generate aberrant transcripts and dysfunctional proteins, potentially triggering type I interferon responses or creating neoantigens that drive autoantibody production. This is particularly relevant for LDGs, a subset already characterized by proinflammatory activity and spontaneous, dysregulated NET formation ( 32 ). Among the altered splicing events, mutually exclusive exon usage, a highly conserved and tightly regulated mechanism across tissues ( 47 – 49 ), was particularly enriched in SLE LDGs. For example, GBP5 , which encodes an interferon-inducible GTPase involved in inflammatory responses and lupus nephritis progression in mice ( 50 , 51 ) exhibited both mutually exclusive exon usage and exon skipping in lupus neutrophils compared to healthy controls. GBP5+ neutrophils with elevated interferon signatures have also been identified in inflamed human tissues, such as nasal polyps, indicating broader relevance beyond lupus ( 52 ). In NDGs, splicing disruptions were detected in SRPK1 , a splicing kinase involved in neutrophil development and linked to myelodysplastic syndromes ( 53 ). Additionally, several MAPK family members, many of which contain U12 introns and play essential roles in stress signaling, were affected (Supplementary Data S1-4). Importantly, U12 intron-containing genes are generally less efficiently spliced due to the instability of U6atac, a key snRNA in the minor spliceosome ( 54 , 55 ). Notably, MAPK14 (p38), itself U12-regulated, is critical for stabilizing U6atac under stress. This suggests a feedback loop where impaired p38 expression may further destabilize U12 splicing, compounding splicing dysfunction in SLE neutrophils. This mechanism may also operate in other immune cells, as indicated by the U12 splicing abnormalities detected in SLE PBMCs. (Supplementary Data S4). Additional support for a splicing-driven dysfunction in neutrophils comes from altered splicing in DOCK2 , a Rac activator essential for actin cytoskeleton dynamics and oxidative burst ( 56 ). Combined with multiple splicing events detected in Nox components beyond CYBA , these findings offer a putative mechanistic explanation for the reduced respiratory burst observed in SLE LDGs. Our data reinforce the distinctiveness of the LDG subset and implicate it in SLE pathogenesis through both compromised Nox function and pervasive U12 splicing defects. This aligns with genetic studies linking polymorphisms in Nox genes to elevated SLE risk ( 57 – 59 ). Importantly, U12 splicing anomalies in LDGs associate with disease activity and specific autoantibody profiles, underscoring a possible role for splicing errors in generating autoantigens and amplifying autoimmune responses in SLE. While we observed a trend toward reduced CYBB protein levels in SLE LDGs compared to NDGs and healthy controls, this difference did not reach statistical significance. Nevertheless, this pattern complements the broader Nox dysregulation observed. CYBB and CYBA (p22phox) are critical Nox subunits responsible for ROS generation during the oxidative burst. The concurrent reduction of these components in LDGs suggests a coordinated impairment of complex assembly or stability, potentially contributing to the diminished oxidative burst capacity. Since CYBB lacks a U12-type intron, its reduced expression likely reflects additional mechanisms beyond minor spliceosome dysfunction, such as altered transcriptional regulation, enhanced protein degradation, retrotransposable genetic elements causing mutations, or LDG-specific epigenetic modifications ( 18 , 60 , 61 ). Collectively, these findings highlight multifactorial defects in the oxidative machinery of LDGs that may underlie their heightened inflammatory and pathogenic potential. A plausible driver of the widespread splicing abnormalities, especially those involving U12 introns, is chronic cellular stress stemming from persistent systemic inflammation and oxidative imbalance. LDGs are known to produce elevated mitochondrial ROS ( 33 ), which could impair spliceosome components or their regulatory kinases. The intrinsic instability of U6atac snRNA further sensitizes U12 splicing to stress-induced disruption. Genetic factors, such as SNPs in spliceosome-related genes or regulatory kinases like SRPK1 and MAPK14/p38, may exacerbate splicing defects ( 62 , 63 ). Moreover, autoantibodies targeting spliceosomal proteins, commonly present in SLE patients, might directly impair splicing machinery, increasing transcriptomic noise and producing aberrant RNA species. These combined disruptions could promote novel autoantigen formation, perpetuating autoimmunity and disease progression. Although both LDGs and NDGs from SLE patients exhibited increased retention of U12-type introns, the decrease in CYBA expression was specific to LDGs. This selective downregulation suggests that U12 splicing defects alone do not fully explain altered gene expression in SLE neutrophils. Instead, cell-intrinsic factors unique to LDGs, such as altered maturation, epigenetic remodeling, and activation states ( 32 ), likely influence transcript stability, translation efficiency, or protein turnover. For example, CYBA transcripts may be more susceptible to degradation of mis-spliced RNA or differential regulation by RNA-binding proteins within LDGs. These findings support the concept that the functional impact of U12 splicing dysregulation is context-dependent, preferentially disrupting gene networks critical for oxidative burst and immune regulation in distinct neutrophil subsets. While our study provides novel insights into U12 intron splicing defects and neutrophil dysfunction in SLE, several limitations warrant consideration. First, the sample size was modest, which limits statistical power and the robustness of some clinical correlations. This includes uneven group sizes in RNA-seq comparisons, which increase the risk of false discovery. Larger, well-stratified cohorts will be required to confirm these preliminary findings and extend them to broader patient populations, including individuals with CGD. Second, while transcriptomic analysis revealed widespread splicing changes, functional validation of specific splice variants, such as their effects on protein expression and enzymatic activity, was only partially addressed. Future studies should directly define how these splicing alterations affect neutrophil biology and immune responses. Finally, although we observed correlations with clinical features, causal relationships remain unproven, and our focus on neutrophils does not exclude the possibility that U12 splicing defects may extend to other immune cell types in SLE, as suggested by PBMC analyses. In summary, our findings highlight disrupted U12 intron splicing as a previously underappreciated contributor to neutrophil dysfunction in SLE, particularly within the pathogenic LDG subset. This study broadens our understanding of lupus pathogenesis and opens new avenues for developing therapeutic strategies that target RNA splicing pathways. Author Contributions All authors were involved in revising the manuscript for important intellectual content, and all authors approved the final version to be published. Dr Blanco wrote the original version of this manuscript. Drs. Kaplan and Sun have full access to all the data in the study and the manuscript responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design Blanco, Sun, Kaplan. Acquisition of data Blanco, Remi, Liu, Wang, Carlucci, Jackson, Carmona-Rivera, Hasni, Hafner, Sun. Analysis and of data Blanco, Remi, Sun. Acknowledgment This work utilized the computational resources of the NIH HPC Biowulf cluster ( https://hpc.nih.gov ). This study was supported by the Intramural Research Program at NIAMS (ZIA AR041199). This research was supported [in part] by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Funder Information Declared National Institute of Arthritis and Musculoskeletal and Skin Diseases, https://ror.org/006zn3t30 , ZIAAR041199 References 1. ↵ Rodriguez MA , Blasini AM . Just Autoimmunity? The Role of the Innate Immune Response in Lupus. JCR: Journal of Clinical Rheumatology . 2025 ; 31 ( 2 ): 71 – 7 . OpenUrl PubMed 2. ↵ Ast G , Goldblatt D , Waisman A , Sperling R , Mozes E , Sperling J . An autoantibody derived from mice with experimental systemic lupus erythematosus is directed against the essential splicing factor SF53/4--a possible role for large nuclear ribonucleoprotein particles in autoimmune disorders . International Immunology . 1994 ; 6 ( 8 ): 1097 – 105 . OpenUrl CrossRef PubMed Web of Science 3. Kuwana M , Gil-Vila A , Selva-O’Callaghan A . Role of autoantibodies in the diagnosis and prognosis of interstitial lung disease in autoimmune rheumatic disorders . Therapeutic Advances in Musculoskeletal Disease . 2021 ; 13 : 1759720X211032457 . OpenUrl PubMed 4. Dumortier H , Monneaux F , Jahn-Schmid B , Briand J-P , Skriner K , Cohen PL , et al. B and T Cell Responses to the Spliceosomal Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in Normal and Lupus Mice . The Journal of Immunology . 2000 ; 165 ( 4 ): 2297 – 305 . OpenUrl PubMed 5. Monneaux F , Dumortier H , Steiner G , Briand J-P , Muller S . Murine models of systemic lupus erythematosus: B and T cell responses to spliceosomal ribonucleoproteins in MRL/Faslpr and (NZB × NZW)F1 lupus mice . International Immunology . 2001 ; 13 ( 9 ): 1155 – 63 . OpenUrl CrossRef PubMed Web of Science 6. Stochmal A , Czuwara J , Trojanowska M , Rudnicka L . Antinuclear Antibodies in Systemic Sclerosis: an Update . Clinical Reviews in Allergy & Immunology . 2020 ; 58 ( 1 ): 40 – 51 . OpenUrl PubMed 7. Callejas-Moraga EL , Guillén-Del-Castillo A , Perurena-Prieto J , Sanz-Martínez MT , Fonollosa-Pla V , Lorite-Gomez K , et al. Anti-RNPC-3 antibody predicts poor prognosis in patients with interstitial lung disease associated to systemic sclerosis . Rheumatology . 2021 ; 61 ( 1 ): 154 – 62 . OpenUrl PubMed 8. Fritzler MJ , Bentow C , Beretta L , Palterer B , Perurena-Prieto J , Sanz-Martínez MT , et al. Anti-U11/U12 Antibodies as a Rare but Important Biomarker in Patients with Systemic Sclerosis: A Narrative Review . Diagnostics . 2023 ; 13 ( 7 ): 1257 . OpenUrl PubMed 9. ↵ McMahan ZH , Domsic RT , Zhu L , Medsger TA , Casciola-Rosen L , Shah AA . Anti–RNPC-3 (U11/U12) Antibodies in Systemic Sclerosis in Patients With Moderate-to-Severe Gastrointestinal Dysmotility . Arthritis Care & Research . 2019 ; 71 ( 9 ): 1164 – 70 . OpenUrl PubMed 10. ↵ Ueda MT , Inamo J , Miya F , Shimada M , Yamaguchi K , Kochi Y . Functional and dynamic profiling of transcript isoforms reveals essential roles of alternative splicing in interferon response . Cell Genom . 2024 ; 4 ( 10 ): 100654 . OpenUrl PubMed 11. Wilkinson ME , Charenton C , Nagai K . RNA Splicing by the Spliceosome . Annual Review of Biochemistry . 2020 ; 89 (Volume 89, 2020): 359 – 88 . OpenUrl CrossRef PubMed 12. RNA splicing — a central layer of gene regulation . Nature Reviews Genetics . 2025 ; 26 ( 6 ): 369 – 70 . OpenUrl PubMed 13. Fast NM . Intron splicing: U12 spliceosomal introns not so ‘minor’ after all . Current Biology . 2021 ; 31 ( 14 ): R912 – R4 . OpenUrl PubMed 14. Norppa AJ , Shcherbii MV , Frilander MJ . Connecting genotype and phenotype in minor spliceosome diseases . Rna . 2025 . 15. Maik-Rachline G , Wortzel I , Seger R . Alternative Splicing of MAPKs in the Regulation of Signaling Specificity . Cells . 2021 ; 10 ( 12 ). 16. Scotti MM , Swanson MS . RNA mis-splicing in disease . Nat Rev Genet . 2016 ; 17 ( 1 ): 19 – 32 . OpenUrl CrossRef PubMed 17. Wang J , Ruan GX , Li Y , Xiao X , Zhu Z , Chen W , et al. Minor Splicing Factor RNPC3 Is Essential for the Germinal Center B Cell Response . Eur J Immunol . 2025 ; 55 ( 4 ): e202451508 . OpenUrl PubMed 18. ↵ Katsuyama T , Moulton VR . Splicing factor SRSF1 is indispensable for regulatory T cell homeostasis and function . Cell Reports . 2021 ; 36 ( 1 ): 109339 . OpenUrl PubMed 19. de Wolf B , Oghabian A , Akinyi MV , Hanks S , Tromer EC , van Hooff JJE , et al. Chromosomal instability by mutations in the novel minor spliceosome component CENATAC . The EMBO Journal . 2021 ; 40 ( 14 ): e106536 . OpenUrl CrossRef PubMed 20. Li L , Ding Z , Pang T-L , Zhang B , Li C-H , Liang A-M , et al. Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila . Nature Communications . 2020 ; 11 ( 1 ): 5608 . OpenUrl PubMed 21. Madan V , Cao Z , Teoh WW , Dakle P , Han L , Shyamsunder P , et al. ZRSR1 co-operates with ZRSR2 in regulating splicing of U12-type introns in murine hematopoietic cells . Haematologica . 2022 ; 107 ( 3 ): 680 – 9 . OpenUrl CrossRef PubMed 22. Madan V , Kanojia D , Li J , Okamoto R , Sato-Otsubo A , Kohlmann A , et al. Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome . Nat Commun . 2015 ; 6 . 23. Togami K , Chung SS , Madan V , Booth CAG , Kenyon CM , Cabal-Hierro L , et al. Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis . Cancer Discovery . 2022 ; 12 ( 2 ): 522 – 41 . OpenUrl Abstract / FREE Full Text 24. ↵ Osman EY , Van Alstyne M , Yen P-F , Lotti F , Feng Z , Ling KKY , et al. Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons . JCI Insight . 2020 ; 5 ( 12 ). 25. ↵ Naor D , Nedvetzki S , Golan I , Melnik L , Faitelson Y . CD44 in cancer . Crit Rev Clin Lab Sci . 2002 ; 39 ( 6 ): 527 – 79 . OpenUrl CrossRef PubMed Web of Science 26. Moulton VR , Tsokos GC . Alternative splicing factor/splicing factor 2 regulates the expression of the zeta subunit of the human T cell receptor-associated CD3 complex . J Biol Chem . 2010 ; 285 ( 17 ): 12490 – 6 . OpenUrl Abstract / FREE Full Text 27. Zisman E , Stern O , Lewis R , Tzaban S , Eisenhaure T , Henrickson SE , et al. Unveiling alternative splicing dynamics in activated T lymphocytes and their implications for immune checkpoint blockade efficacy . iScience . 2025 ; 28 ( 5 ): 112434 . OpenUrl PubMed 28. Molina CA , Foulkes NS , Lalli E , Sassone-Corsi P . Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor . Cell . 1993 ; 75 ( 5 ): 875 – 86 . OpenUrl CrossRef PubMed Web of Science 29. Agrebi N , Ben-Mustapha I , Matoussi N , Dhouib N , Ben-Ali M , Mekki N , et al. Rare splicing defects of FAS underly severe recessive autoimmune lymphoproliferative syndrome . Clin Immunol . 2017 ; 183 : 17 – 23 . OpenUrl PubMed 30. ↵ Melard P , Idrissi Y , Andrique L , Poglio S , Prochazkova-Carlotti M , Berhouet S , et al. Molecular alterations and tumor suppressive function of the DUSP22 (Dual Specificity Phosphatase 22) gene in peripheral T-cell lymphoma subtypes . Oncotarget . 2016 ; 7 ( 42 ): 68734 – 48 . OpenUrl PubMed 31. ↵ Wigerblad G , Kaplan MJ . Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases . Nature Reviews Immunology . 2023 ; 23 ( 5 ): 274 – 88 . OpenUrl CrossRef PubMed 32. ↵ Carmona-Rivera C , Kaplan MJ . Low-density granulocytes in systemic autoimmunity and autoinflammation . Immunol Rev . 2023 ; 314 ( 1 ): 313 – 25 . OpenUrl CrossRef PubMed 33. ↵ Lood C , Blanco LP , Purmalek MM , Carmona-Rivera C , De Ravin SS , Smith CK , et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease . Nature medicine . 2016 ; 22 ( 2 ): 146 – 53 . OpenUrl CrossRef PubMed 34. ↵ Ambasta RK , Kumar P , Griendling KK , Schmidt HH , Busse R , Brandes RP . Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase . J Biol Chem . 2004 ; 279 ( 44 ): 45935 – 41 . OpenUrl Abstract / FREE Full Text 35. ↵ Brandes RP , Schroder K . NOXious phosphorylation: Smooth muscle reactive oxygen species production is facilitated by direct activation of the NADPH oxidase Nox1 . Circ Res . 2014 ; 115 ( 11 ): 898 – 900 . OpenUrl FREE Full Text 36. ↵ Kelkka T , Kienhofer D , Hoffmann M , Linja M , Wing K , Sareila O , et al. Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature . Antioxid Redox Signal . 2014 ; 21 ( 16 ): 2231 – 45 . OpenUrl CrossRef PubMed 37. ↵ Blanco LP , Pedersen HL , Wang X , Lightfoot YL , Seto N , Carmona-Rivera C , et al. Improved Mitochondrial Metabolism and Reduced Inflammation Following Attenuation of Murine Lupus With Coenzyme Q10 Analog Idebenone . Arthritis Rheumatol . 2020 ; 72 ( 3 ): 454 – 64 . OpenUrl PubMed 38. ↵ Hochberg MC . Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus . Arthritis Rheum . 1997 ; 40 ( 9 ): 1725 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Carmona-Rivera C , Simeonov DR , Cardillo ND , Gahl WA , Cadilla CL . A divalent interaction between HPS1 and HPS4 is required for the formation of the biogenesis of lysosome-related organelle complex-3 (BLOC-3) . Biochim Biophys Acta . 2013 ; 1833 ( 3 ): 468 – 78 . OpenUrl 40. ↵ Shen S , Park JW , Lu Z-x , Lin L , Henry MD , Wu YN , et al. rMATS: Robust and flexible detection of differential alternative splicing from replicate RNA-Seq data . Proceedings of the National Academy of Sciences . 2014 ; 111 ( 51 ): E5593 – E601 . OpenUrl Abstract / FREE Full Text 41. ↵ Gnatowski ER , Jurmain JL , Dozmorov MG , Wolstenholme JT , Miles MF . Ninein, a candidate gene for ethanol anxiolysis, shows complex exon-specific expression and alternative splicing differences between C57BL/6J and DBA/2J mice . Front Genet . 2024 ; 15 : 1455616 . 42. ↵ Chen EY , Tan CM , Kou Y , Duan Q , Wang Z , Meirelles GV , et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool . BMC Bioinformatics . 2013 ; 14 : 128 . 43. Kuleshov MV , Jones MR , Rouillard AD , Fernandez NF , Duan Q , Wang Z , et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update . Nucleic Acids Res . 2016 ; 44 ( W1 ): W90 – 7 . OpenUrl CrossRef PubMed 44. ↵ Xie Z , Bailey A , Kuleshov MV , Clarke DJB , Evangelista JE , Jenkins SL , et al. Gene Set Knowledge Discovery with Enrichr . Current Protocols . 2021 ; 1 ( 3 ): e90 . OpenUrl CrossRef 45. ↵ Dinauer MC . Chronic Granulomatous Disease and Other Disorders of Phagocyte Function . ASH Education Program Book . 2005 ; 2005 ( 1 ): 89 – 95 . OpenUrl 46. ↵ Cheng Q , Chen M , Chen X , Chen X , Jiang H , Wu H , et al. Novel Long Non-coding RNA Expression Profile of Peripheral Blood Mononuclear Cells Reveals Potential Biomarkers and Regulatory Mechanisms in Systemic Lupus Erythematosus . Front Cell Dev Biol . 2021 ; 9 : 639321 . 47. ↵ Hatje K , Rahman RU , Vidal RO , Simm D , Hammesfahr B , Bansal V , et al. The landscape of human mutually exclusive splicing . Mol Syst Biol . 2017 ; 13 ( 12 ): 959 . OpenUrl Abstract / FREE Full Text 48. Lam SD , Babu MM , Lees J , Orengo CA . Biological impact of mutually exclusive exon switching . PLoS Comput Biol . 2021 ; 17 ( 3 ): e1008708 . OpenUrl PubMed 49. ↵ Li S , Lv H , Zhang R , Li J , Chen Z , Yang N , et al. Aging-related alternative splicing drive neoantigen emergence revealed by transcriptome analysis of 1,255 human blood samples . Front Aging . 2025 ; 6 : 1575862 . 50. ↵ Liu N , Gao Y , Liu Y , Liu D . GBP5 Inhibition Ameliorates the Progression of Lupus Nephritis by Suppressing NLRP3 Inflammasome Activation . Immunol Invest . 2023 ; 52 ( 1 ): 52 – 66 . OpenUrl PubMed 51. ↵ Ren F , Li J , Ni X , Yuan H , Yan W . Knockdown of GBP5 alleviates renal damage caused by psoriasis by regulating NF-κB/STAT3 pathway . Allergol Immunopathol (Madr ). 2024 ; 52 ( 6 ): 117 – 27 . OpenUrl PubMed 52. ↵ Zhang C , Zhang Q , Chen J , Li H , Cheng F , Wang Y , et al. Neutrophils in nasal polyps exhibit transcriptional adaptation and proinflammatory roles that depend on local polyp milieu . JCI Insight . 2024 ; 9 ( 22 ). 53. ↵ Tzelepis K , De Braekeleer E , Aspris D , Barbieri I , Vijayabaskar MS , Liu W-H , et al. SRPK1 maintains acute myeloid leukemia through effects on isoform usage of epigenetic regulators including BRD4 . Nature Communications . 2018 ; 9 ( 1 ): 5378 . OpenUrl PubMed 54. ↵ Patel AA , McCarthy M , Steitz JA . The splicing of U12-type introns can be a rate-limiting step in gene expression . The EMBO Journal . 2002 ; 21 ( 14 ): 3804 – 15 . OpenUrl Abstract / FREE Full Text 55. ↵ Younis I , Dittmar K , Wang W , Foley SW , Berg MG , Hu KY , et al. Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA . eLife . 2013 ; 2 : e00780 . OpenUrl CrossRef PubMed 56. ↵ Moens L , Gouwy M , Bosch B , Pastukhov O , Nieto-Patlàn A , Siler U , et al. Human DOCK2 Deficiency: Report of a Novel Mutation and Evidence for Neutrophil Dysfunction . J Clin Immunol . 2019 ; 39 ( 3 ): 298 – 308 . OpenUrl CrossRef PubMed 57. ↵ Jacob CO , Eisenstein M , Dinauer MC , Ming W , Liu Q , John S , et al. Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase . Proceedings of the National Academy of Sciences . 2012 ; 109 ( 2 ): E59 – E67 . OpenUrl Abstract / FREE Full Text 58. Kim-Howard X , Sun C , Molineros JE , Maiti AK , Chandru H , Adler A , et al. Allelic heterogeneity in NCF2 associated with systemic lupus erythematosus (SLE) susceptibility across four ethnic populations . Human Molecular Genetics . 2014 ; 23 ( 6 ): 1656 – 68 . OpenUrl CrossRef PubMed Web of Science 59. ↵ Armstrong DL , Eisenstein M , Zidovetzki R , Jacob CO . Systemic Lupus Erythematosus-associated Neutrophil Cytosolic Factor 2 mutation affects the structure of the NADPH oxidase complex . Journal of Biological Chemistry . 2015 . 60. ↵ Roos D , de Boer M . Retrotransposable genetic elements causing neutrophil defects . Eur J Clin Invest . 2018 ; 48 Suppl 2 : e12953 . OpenUrl PubMed 61. ↵ Mistry P , Nakabo S , O’Neil L , Goel RR , Jiang K , Carmona-Rivera C , et al. Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus . Proc Natl Acad Sci U S A . 2019 ; 116 ( 50 ): 25222 – 8 . OpenUrl Abstract / FREE Full Text 62. ↵ Cao M , Yan J , Ding Y , Zhang Y , Sun Y , Jiang G , et al. The potential impact of RNA splicing abnormalities on immune regulation in endometrial cancer . Cell Death Dis . 2025 ; 16 ( 1 ): 148 . OpenUrl PubMed 63. ↵ Douglas KB , Windels DC , Zhao J , Gadeliya AV , Wu H , Kaufman KM , et al. Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing . Genes Immun . 2009 ; 10 ( 5 ): 457 – 69 . OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted November 07, 2025. Download PDF Supplementary Material 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 Dysregulation of U12-Type Splicing in Lupus Neutrophils 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 Dysregulation of U12-Type Splicing in Lupus Neutrophils Luz P. Blanco , Binod Regmi , Carmelo Carmona-Rivera , Yudong Liu , Xiantao Wang , Philip M. Carlucci , Monica M. Jackson , Zerai Manna , Sarfaraz Hasni , Markus Hafner , Hong-Wei Sun , Mariana J. Kaplan bioRxiv 2025.11.06.686965; doi: https://doi.org/10.1101/2025.11.06.686965 Share This Article: Copy Citation Tools Dysregulation of U12-Type Splicing in Lupus Neutrophils Luz P. Blanco , Binod Regmi , Carmelo Carmona-Rivera , Yudong Liu , Xiantao Wang , Philip M. Carlucci , Monica M. Jackson , Zerai Manna , Sarfaraz Hasni , Markus Hafner , Hong-Wei Sun , Mariana J. 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