Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A Splicing Complex

Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A Splicing Complex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A Splicing Complex Zhengfan Zheng, Suisui Guo, Yanxia Rao, Man-Ning Hui, May Pui Lai Cheung, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2603667/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Emerging evidence reveals the crucial role of ubiquitous splicing factors in neural crest (NC) development, which is highly susceptible to splicing dysregulation. How these factors integrate with the NC gene regulatory network (GRN) and contribute to the increased vulnerability of NC to splicing perturbations are not well understood. Here, we show that NC-specific DLC1, in association with the SF3B1-PHF5A splicing complex, are crucial for determining avian trunk NC cell fate by regulating the splicing of pre-mRNA for NC specifiers SOX9 and SNAI2 rather than their upstream regulators BMP4, WNT1, and PAX7 in the GRN. Mechanistically, SF3B1-PHF5A are associated with the branch point adenosine (BPA) of all the factors’ introns, while DLC1 binds specifically to the BPA at the introns of SOX9 and SNAI2, thereby determining the functional specificity of the SF3B1-PHF5A complex in NC specification. Moreover, DLC1 enhances the susceptibility of NC cells to the splicing modulator pladienolide B by reducing the binding capacity of the SF3B1-PHF5A splicing complex to the shorter length of both SOX9 intron 2 and SNAI2 intron 1, which have weaker polypyrimidine tract 3’ of the BPA, resulting in intron retention and loss of NC progenitors. Our data highlight the critical role of the DLC1-SF3B1-PHF5A splicing complex in determining trunk NC cell fate and conferring its increased susceptibility to splicing perturbation. Biological sciences/Developmental biology Biological sciences/Molecular biology/Transcription Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations There is NO Competing Interest. Table 1 is available in the Supplementary Files section. Supplementary Files SupplementaryFiglegends.pdf Supplementary Materials DataS1.xlsx Dataset 1 DataS2.xlsx Dataset 2 DatasetS120240406.xlsx Dataset S1 DatasetS220240406.xlsx Dataset S2 DatasetS320240406.xlsx Dataset S3 Table1.png Cite Share Download PDF Status: Published Journal Publication published 21 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2603667","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":300579935,"identity":"81d52b91-4273-400e-a046-cd2af689cb5e","order_by":0,"name":"Zhengfan Zheng","email":"","orcid":"","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Zhengfan","middleName":"","lastName":"Zheng","suffix":""},{"id":300579937,"identity":"222ec306-7295-4343-8587-09be53d9b368","order_by":1,"name":"Suisui Guo","email":"","orcid":"","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Suisui","middleName":"","lastName":"Guo","suffix":""},{"id":300579939,"identity":"e8658175-b163-4ea1-bada-3764966b40a1","order_by":2,"name":"Yanxia Rao","email":"","orcid":"https://orcid.org/0000-0001-5292-2056","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai 21108, China","correspondingAuthor":false,"prefix":"","firstName":"Yanxia","middleName":"","lastName":"Rao","suffix":""},{"id":300579941,"identity":"3f24392e-e8cb-455f-80bf-64528cf0ff4e","order_by":3,"name":"Man-Ning Hui","email":"","orcid":"","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Department of Obstetrics and Gynaecology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Man-Ning","middleName":"","lastName":"Hui","suffix":""},{"id":300579942,"identity":"22eb1f52-15d0-443d-92b9-e4af2f2fc5db","order_by":4,"name":"May Pui Lai Cheung","email":"","orcid":"","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"May","middleName":"Pui Lai","lastName":"Cheung","suffix":""},{"id":300579943,"identity":"558092fb-21b9-43b9-b8f1-ae57760725a1","order_by":5,"name":"Hoi Yau Tam","email":"","orcid":"","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Hoi","middleName":"Yau","lastName":"Tam","suffix":""},{"id":300579944,"identity":"b0a7e4b3-16a0-44eb-92d7-aaa9bc9fd726","order_by":6,"name":"Kelvin K. W. Wong","email":"","orcid":"","institution":"Centre for PanorOmic Sciences Proteomics and Metabolomics Core Facility, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Kelvin","middleName":"K. W.","lastName":"Wong","suffix":""},{"id":300579945,"identity":"aeec5818-ae96-4169-8ec4-49071f49b45f","order_by":7,"name":"Rakesh Sharma","email":"","orcid":"","institution":"Centre for PanorOmic Sciences Proteomics and Metabolomics Core Facility, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"","lastName":"Sharma","suffix":""},{"id":300579947,"identity":"40264c4b-e933-497c-85ce-643b323fe4c3","order_by":8,"name":"Jessica Aijia Liu","email":"","orcid":"","institution":"Department of Neuroscience, Tat Chee Avenue, City University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"Aijia","lastName":"Liu","suffix":""},{"id":300579932,"identity":"a28fc1ad-d6c6-4a1d-b78f-4ed776ce3337","order_by":9,"name":"Martin Cheung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYFACHjApxy/BxsDA2JBAvBZjyRmkaknccINYLbrtZw8+5t1Rm7j5dluaBOOONMJazM7kJRvznjluvO3OsWMSjGdyiNByg8dMmrftmOy2G+ltEoxtFURpMf8N1MK4eQYJWsyYedtqFDdIpAEd1kaMw87kGEvObTtgLHEjLdkisY0Y7x8/Y/jhbVudHP+MNMMbH9uSCWuBgsMQKoFoDQwMdSSoHQWjYBSMghEHAMqLOy+Yo/3uAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3471-8534","institution":"School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China","correspondingAuthor":true,"prefix":"","firstName":"Martin","middleName":"","lastName":"Cheung","suffix":""}],"badges":[],"createdAt":"2023-02-19 07:10:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2603667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2603667/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-62003-6","type":"published","date":"2025-07-21T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56213690,"identity":"0e3d8536-a118-4251-84c0-4eb24745b122","added_by":"auto","created_at":"2024-05-10 02:19:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2437422,"visible":true,"origin":"","legend":"\u003cp\u003eDLC1 is required for NC specification through the regulation of SOX9 and SNAI2 pre-mRNA splicing. a Hybridization chain reaction (HCR) revealed the co-localization of DLC1, SOX9, and SNAI2 mRNA in premigratory NCCs. b Schematic diagram of gRNA 1 and gRNA 2 oligos targeting different regions of DLC1 exon 9. c Experimental workflow. d Western blot showing loss of DLC1 expression in both gRNA treatment groups compared to control gRNA (Ctrl gRNA). e Reduced expression of SOX9, SNAI2, and FOXD3 in dorsal neural tubes transfected with DLC1 gRNA shown by in situ hybridization (ISH) (blue arrowheads). Black arrows indicate unaltered and restored expression of SOX9, SNAI2 and FOXD3 in Ctrl gRNA and rescue treatments, respectively. Scale bar, 50 μm (n=3). f to h RT-qPCR demonstrating reduced expression levels of SOX9, SNAI2 and FOXD3 in DLC1-depleted cells compared to Ctrl gRNA (n=5). i Immunofluorescence (IF) illustrating nuclear localization of ectopic V5-DLC1 (open arrowheads) in the dorsal neural tube. j Electroporation of DLC1 gRNA1 leading to reduced SOX9 expression (white open arrowheads) and upregulated SOX2 expression (white solid arrowheads) in the dorsal neural tube. Dotted lines outline the neural tube. Scale bar, 50 μm (n=3 per group). k Whole mount IF and the transverse sections reveal reduced expression of HNK-1 (white open arrowheads) in DLC1 gRNA 1 transfected side. White dotted lines in the whole mount IF indicate the axial level of transverse sections. Scale bar, 100 μm (n=3). l Western blot showing unaltered expression of RNA Pol Ⅱ- pSer5 and -pSer2 in embryos treated with DLC1 gRNA 1 and gRNA 2 compared to Ctrl gRNA (n=10 per group). Intron retention-qPCR (IR-qPCR) analysis indicated increased levels of SOX9 introns 1/2 (m) and SNAI2 introns 1/3 (o) in embryos treated with DLC1 gRNA1 compared to Ctrl gRNA (n=5). Schematic diagram illustrating intron-retained SOX9 (n) and SNAI2 (p) pre-mRNA in DLC1-depleted cells. Arrows indicate primer pairs flanking the branch point adenosine (A) in each intron for qPCR analysis. q HCR showing the detection of retained intron1/2 of SOX9 and introns 1/3 of SNAI2 in dorsal neural tubes transfected with DLC1 gRNA1, while no signal was detected in Ctrl gRNA (n=3). Dotted lines outline the transverse neural tubes where the “+” indicates the transfected side. Scale bar, 50 μm. Mean ± SD. **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/9593d9bd98220480a1446fed.png"},{"id":56214285,"identity":"29241f1e-3969-49db-ae50-63e88b420ae9","added_by":"auto","created_at":"2024-05-10 02:28:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2617753,"visible":true,"origin":"","legend":"\u003cp\u003ePHF5A, an interacting factor of DLC1, plays a crucial role 903 in NC specification by regulating the splicing of SOX9 and SNAI2 pre-mRNA. a Interactome of DLC1-associated splicing factors. Proteins of interest, PHF5A and SF3B1, are color-coded in brown. Immunoprecipitation (IP) validated the association of endogenous DLC1 with PHF5A and SF3B1. b Interactome of PHF5A showing splicing factors in the SF3B complex (green color). IP confirmed the association of endogenous SF3B1 with PHF5A. c Schematic diagram of gRNA 1 and gRNA 2 oligos targeting exon 3 and exon 4 of PHF5A. Western blot showing abolishment of PHF5A expression in embryos treated with PHF5A gRNA compared to embryos treated with Ctrl gRNA (n=10). IF and ISH show reduced expression (white open arrowheads) of SOX9 (d), SNAI2 (f), and FOXD3 (h) in PHF5A-ablated cells compared to Ctrl gRNA. e, g and i qRT-PCR confirmed the reduction of these genes. The expression of NC specifier genes was restored by pCIG-HA-PHF5A overexpression. Black arrows indicate unaltered expression of NC specifier genes in Ctrl gRNA embryos and restoration of NC specifier genes expression in the rescue groups. Scale bar, 50 μm (n=3 per group). j SOX9 expression (white open arrowheads) was reduced in PHF5A-depleted NCCs where SOX2 expression was upregulated (white solid arrowheads). Dotted lines outline the neural tube. Scale bar, 50 μm (n=3 per group). k HNK-1 expression was reduced (white open arrowheads) in the PHF5A gRNA-transfected side compared to the untransfected side and embryo treated with Ctrl gRNA. l Expression levels of RNA PolⅡ-pSer5 and -pSer2 remained unaltered in PHF5A KO embryos compared to Ctrl gRNA (n=10). IR-qPCR analysis showed increased levels of SOX9 introns 1/2 (m) and SNAI2 introns 1/3 (o) in embryos treated with PHF5A gRNA1 compared to Ctrl gRNA-treated embryos (n=5). Schematic diagram showing intron-retained SOX9 and SNAI2 pre-mRNA in PHF5A-ablated cells. Arrows indicate primer pairs for the detection of retained introns of SOX9 (n) and SNAI2 (p). q HCR showing detection of retained introns of SOX9 and SNAI2 in embryos transfected with PHF5A gRNA. Dotted lines outline the transverse neural tubes where the “+” indicates the transfected side. Scale bar: 50 μm. Mean ± SD. **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/7730d9ca6020a6c2f82d1dcf.png"},{"id":56212639,"identity":"09eec850-19c6-4b2f-acdc-37434c11a309","added_by":"auto","created_at":"2024-05-10 02:11:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":695622,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic profiling reveals increased intron retention of NC specifier genes in embryos lacking DLC1, PHF5A, or SF3B1 in the neural tubes. Elevated intron expression levels of SOX9 (a, b), SNAI2 (c, d), ID1 (e), TWIST1 (f), and MYC (g) were detected in the absence of DLC1, PHF5A, or SF3B1 compared to Ctrl gRNA. Intron levels of BMP4 (h), WNT1 (i, j), PAX7 (k, l, m), and MYB (934 n) were unaltered in embryos lacking DLC1, PHF5A, or SF3B1 in the neural tubes.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/fc72dad7bc8bd65d9f6c8805.png"},{"id":56212636,"identity":"faa831c6-415b-4a59-98e6-37f88dd5ac81","added_by":"auto","created_at":"2024-05-10 02:11:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":368721,"visible":true,"origin":"","legend":"\u003cp\u003eDLC1 is required for the binding of PHF5A and SF3B1 to the introns of NC specifier genes. a, b Schematic diagram shows primer pairs flanking the BPA of SOX9 (a) and SNAI2 (b) introns for RNA-immunoprecipitation qPCR (RIP-qPCR). c to h Graphs show the binding capacity of endogenous PHF5A (c, f), SF3B1 (d, g), and DLC1 (e, h) to the BPA of SOX9 introns 1 and 2 as well as SNAI2 introns 1 and 3 (n=30 per group). (i) IP shows that SF3B1 remained associated with PHF5A in the absence of DLC1. j to m RIP-qPCR analysis reveals reduced binding capacity of PHF5A-SF3B1 splicing complex to introns 1 and 2 of SOX9, as well as introns 1 and 3 of SNAI2 in embryos treated with DLC1 gRNA1 compared to Ctrl gRNA (n=30 per group). Mean ± SD. *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001, ****: p \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/4b37052a7d80ef60dd23e80e.png"},{"id":56212643,"identity":"5332abef-201d-46cf-b1d6-3ecaf0cc07ce","added_by":"auto","created_at":"2024-05-10 02:11:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2201342,"visible":true,"origin":"","legend":"\u003cp\u003eSplicing modulator PB specifically reduces the expression levels of NC specifier genes. a Experimental workflow. b Reduced expression of SOX9, SNAI2, and FOXD3 (open arrowheads) in NCCs of embryos treated with PB, whereas expression of these genes in NCCs (black arrows) and somites (So) remained unaltered in embryos treated with PBS (n=9 per treatment). c PB treatment did not alter the pattern of PAX7 expression (indicated by white arrows) and did not lead to apoptosis, as evidenced by the absence of Caspase-3+ cells. d PB treatment did not affect the expression levels of PHF5A, SF3B1, DLC1, and RNA Pol II, nor induce Caspase 3 expression compared to PBS Ctrl. e IP analysis revealed that endogenous SF3B1 remained associated with PHF5A and DLC1 in the presence of PB. f and h qPCR data demonstrated increased levels of SOX9 intron 2 (f) and SNAI2 intron 3 (h) in the PB treatment groups compared to the PBS Ctrl. No retention of SOX9 intron 1 and SNAI2 intron 3 was detected in the PB treatment group (n=5 per treatment). g and i Schematic diagrams illustrate the presence of intron 2-retained SOX9 pre961 mRNA (g) and intron 1-retained SNAI2 (i) pre-mRNA after PB treatment. j The HCR analysis revealed the expression of SOX9 intron 2 and SNAI2 intron 1 in NCCs of embryos treated with PB, as compared to the PBS Ctrl. k-n 6 sections per treatment were used for the quantification of fluorescence intensities. o - r PB treatment reduced the binding of PHF5A and SF3B1 on both SOX9 intron 2 (o, q) and SNAI2 intron 1 (p, r) compared to PBS Ctrl. Scale 965 bar: 50 μm. NT, neural tube. Mean ± SD. ***: p \u0026lt; 0.001, ****: p \u0026lt; 0.0001, ns: no significant difference.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/047ee629ffea1986b5c3e503.png"},{"id":56212311,"identity":"a4698088-115a-4a16-88ad-0c6cfb9ad3f5","added_by":"auto","created_at":"2024-05-10 02:03:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3262293,"visible":true,"origin":"","legend":"\u003cp\u003eDLC1 determines the increased vulnerability of trunk NCCs to PB treatment. a Experimental workflow. b Ectopic DLC1 expression in somites reduced expression of SOX9 (b, c) and SNAI2 (d, e) in a cell-autonomous manner (open arrowheads) in the PB treatment group. Open arrowheads indicate loss of SOX9 and SNAI2 expression in pre-migratory NCCs. There was no alteration of SOX9 (b, c) and SNAI2 (d, e) expression in DLC1-overexpressing somites and in NCCs (black and white arrows) in the PBS treatment group. Scale bar, 50 μm. Yellow dotted boxes outline the magnified view in the rightmost column of each panel. f IP shows the association of ectopic DLC1 with endogenous PHF5A and SF3B1 in somites of embryos treated with PBS Ctrl and PB. g and i Electroporation of DLC1 in somites resulted in increased levels of SOX9 intron 2 and SNAI2 intron 1 in the presence of PB compared to PBS Ctrl (n=5 per group). h and j Schematic diagrams depict the presence of intron 2-retained SOX9 pre-mRNA (h) and intron 1-retained SNAI2 pre-mRNA (j) in DLC1 overexpressing somites of embryos treated with PB. k The HCR analysis revealed the expression of SOX9 intron 2 and SNAI2 intron 1 in DLC1-overexpressing somites (So) of embryos treated with PB, as compared to the PBS Ctrl (n=5). l-o 20 sections per treatment were used for the quantification of fluorescence intensities. NT, neural tube. Scale bar: 50 μm. Mean ± SD. ***: p \u0026lt; 0.001, ****: p \u0026lt; 0.0001, ns: no significant difference.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/1c7dcb06a666636fd61e4918.png"},{"id":56212317,"identity":"315d3f35-a8d0-4958-8aa4-824dda71ec25","added_by":"auto","created_at":"2024-05-10 02:03:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":604766,"visible":true,"origin":"","legend":"\u003cp\u003eThe binding of the DLC1-SF3B1-PHF5A complex to shorter introns of NC specifier genes renders them more susceptible to splicing perturbation by PB a, b Overexpression of DLC1 in somites of embryos treated with PB resulted in reduced binding capacity of PHF5A to SOX9 intron 2 (a) and SNAI2 intron 1 (b) compared to PBS Ctrl. c, d Overexpression of DLC1 in somites of embryos treated with PB led to a reduction in the binding capacity of SF3B1 to SOX9 intron 2 (c) and SNAI2 intron 1 (d) compared to PBS Ctrl. e In vitro translated SF3B1, DLC1, and PHF5A proteins, each fused with an HIS-tagged, failed to bind to the biotinylated SOX9 intron 2 and SNAI2 intron 1 in the presence of PB. However, PB did not alter the ability of these proteins to bind to the biotinylated SOX9 intron 1 and SNAI2 intron 3. f Schematic diagram depicts the regulation of SOX9 and SNAI2 pre-mRNA splicing in prospective NCCs by the ubiquitously expressed SF3B1-PHF5A splicing factors, in association with NCPage specific DLC1. The DLC1-SF3B1-PHF5A splicing complex plays a 996 crucial role in determining NCC fate and makes them more susceptible to the splicing modulator PB. This susceptibility is due to competitive inhibition of BPA recognition by the complex, which occurs in both SOX9 intron 2 and SNAI2 intron 1 due to their shorter length and weak Py tract, resulting in intron retention and loss of NC progenitors. Mean ± SD. **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001, ns: no significant difference.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/cd4fba69873a7fef1adae84a.png"},{"id":87260416,"identity":"318e2abc-a428-43a5-842f-91eacb676143","added_by":"auto","created_at":"2025-07-22 07:06:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2686489,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ZhengetalMainmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1_covered_7b3d9bf6-e84c-4cc2-88fa-b0430be33c0d.pdf"},{"id":56212321,"identity":"ab53f606-c051-4a25-8353-f8c9ab477b24","added_by":"auto","created_at":"2024-05-10 02:03:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9194312,"visible":true,"origin":"","legend":"Supplementary 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02:03:49","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":101243,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.png","url":"https://assets-eu.researchsquare.com/files/rs-2603667/v1/10640ec7c11cb478617dd7c9.png"}],"financialInterests":"\u003cp\u003eThere is \u003cstrong\u003eNO\u003c/strong\u003e Competing Interest.\u003c/p\u003e\n\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A Splicing Complex","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2603667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2603667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Emerging evidence reveals the crucial role of ubiquitous splicing factors in neural crest (NC) development, which is highly susceptible to splicing dysregulation. How these factors integrate with the NC gene regulatory network (GRN) and contribute to the increased vulnerability of NC to splicing perturbations are not well understood. Here, we show that NC-specific DLC1, in association with the SF3B1-PHF5A splicing complex, are crucial for determining avian trunk NC cell fate by regulating the splicing of pre-mRNA for NC specifiers SOX9 and SNAI2 rather than their upstream regulators BMP4, WNT1, and PAX7 in the GRN. Mechanistically, SF3B1-PHF5A are associated with the branch point adenosine (BPA) of all the factors’ introns, while DLC1 binds specifically to the BPA at the introns of SOX9 and SNAI2, thereby determining the functional specificity of the SF3B1-PHF5A complex in NC specification. Moreover, DLC1 enhances the susceptibility of NC cells to the splicing modulator pladienolide B by reducing the binding capacity of the SF3B1-PHF5A splicing complex to the shorter length of both SOX9 intron 2 and SNAI2 intron 1, which have weaker polypyrimidine tract 3’ of the BPA, resulting in intron retention and loss of NC progenitors. Our data highlight the critical role of the DLC1-SF3B1-PHF5A splicing complex in determining trunk NC cell fate and conferring its increased susceptibility to splicing perturbation.","manuscriptTitle":"Determination of Trunk Neural Crest Cell Fate and Susceptibility to Splicing Perturbation by the DLC1-SF3B1-PHF5A Splicing Complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 02:03:31","doi":"10.21203/rs.3.rs-2603667/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5d48f1a6-222e-4821-9d95-1e3060b984ba","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31714931,"name":"Biological sciences/Developmental biology"},{"id":31714932,"name":"Biological sciences/Molecular biology/Transcription"}],"tags":[],"updatedAt":"2025-07-22T07:06:22+00:00","versionOfRecord":{"articleIdentity":"rs-2603667","link":"https://doi.org/10.1038/s41467-025-62003-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-21 04:00:00","publishedOnDateReadable":"July 21st, 2025"},"versionCreatedAt":"2024-05-10 02:03:31","video":"","vorDoi":"10.1038/s41467-025-62003-6","vorDoiUrl":"https://doi.org/10.1038/s41467-025-62003-6","workflowStages":[]},"version":"v1","identity":"rs-2603667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2603667","identity":"rs-2603667","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}