Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells

Nature Cell Biology volume 15, pages 1141–1152 (2013)Cite this article

Subjects

Abstract

Human embryonic stem cells (hESCs) harbour the ability to undergo lineage-specific differentiation into clinically relevant cell types. Transcription factors and epigenetic modifiers are known to play important roles in the maintenance of pluripotency of hESCs. However, little is known about regulation of pluripotency through splicing. In this study, we identify the spliceosome-associated factor SON as a factor essential for the maintenance of hESCs. Depletion of SON in hESCs results in the loss of pluripotency and cell death. Using genome-wide RNA profiling, we identified transcripts that are regulated by SON. Importantly, we confirmed that SON regulates the proper splicing of transcripts encoding for pluripotency regulators such as OCT4, PRDM14, E4F1 and MED24. Furthermore, we show that SON is bound to these transcripts in vivo. In summary, we connect a splicing-regulatory network for accurate transcript production to the maintenance of pluripotency and self-renewal of hESCs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The effects of SON depletion in hESCs.
Figure 2: Rescue of SON depletion phenotype in hESCs.
Figure 3: RNA-seq analysis of transcripts regulated by SON.
Figure 4: SON regulates splicing of small introns with weak splice sites.
Figure 5: SON targets specific introns of transcripts that encode pluripotency regulators.
Figure 6: Unspliced transcripts induced by SON depletion are potentially targeted by the NMD pathway.
Figure 7: SON regulates alternative splicing in hESCs.
Figure 8: SON knockdown induces an isoform switch of PRDM14.

Similar content being viewed by others

Accession codes

Accessions

ArrayExpress

References

  1. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  PubMed  Google Scholar 

  2. Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Wobus, A. M. & Boheler, K. R. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Kimbrel, E. A. & Lu, S. J. Potential clinical applications for human pluripotent stem cell-derived blood components. Stem Cells Int. 2011, 273076 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Chia, N. Y. et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468, 316–320 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Göke, J., Chan, Y. S., Yan, J., Vingron, M. & Ng, H. H. Genome-wide kinase-chromatin interactions reveal the regulatory network of ERK signaling in human embryonic stem cells. Mol. Cell 50, 844–855 (2013).

    Article  PubMed  Google Scholar 

  8. Göke, J. et al. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development. PLoS Comput. Biol. 7, e1002304 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Jiang, J. et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat. Cell Biol. 10, 353–360 (2008).

    Article  PubMed  Google Scholar 

  10. Loh, Y. H. et al. Genomic approaches to deconstruct pluripotency. Annu. Rev. Genom. Hum. Genet. 12, 165–185 (2011).

    Article  CAS  Google Scholar 

  11. Macarthur, B. D., Ma’ayan, A. & Lemischka, I. R. Systems biology of stem cell fate and cellular reprogramming. Nat. Rev. Mol. Cell Biol. 10, 672–681 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Orkin, S. H. & Hochedlinger, K. Chromatin connections to pluripotency and cellular reprogramming. Cell 145, 835–850 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Young, R. A. Control of the embryonic stem cell state. Cell 144, 940–954 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yuan, P. et al. Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells. Genes Dev. 23, 2507–2520 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Khan, I. M. et al. The SON gene encodes a conserved DNA binding protein mapping to human chromosome 21. Ann. Hum. Genet. 58, 25–34 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Sun, C. T. et al. Transcription repression of human hepatitis B virus genes by negative regulatory element-binding protein/SON. J. Biol. Chem. 276, 24059–24067 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Wynn, S. L. et al. Organization and conservation of the GART/SON/DONSON locus in mouse and human genomes. Genomics 68, 57–62 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Saitoh, N. et al. Proteomic analysis of interchromatin granule clusters. Mol. Biol. Cell 15, 3876–3890 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huen, M. S. et al. SON is a spliceosome-associated factor required for mitotic progression. Cell Cycle 9, 2679–2685 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sharma, A., Takata, H., Shibahara, K., Bubulya, A. & Bubulya, P. A. Son is essential for nuclear speckle organization and cell cycle progression. Mol. Biol. Cell 21, 650–663 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ahn, E. Y. et al. SON controls cell-cycle progression by coordinated regulation of RNA splicing. Mol. Cell 42, 185–198 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sharma, A. et al. Son maintains accurate splicing for a subset of human pre-mRNAs. J. Cell Sci. 124, 4286–4298 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ruiz, S. et al. A high proliferation rate is required for cell reprogrammingand maintenance of human embryonic stem cell identity. Curr. Biol. 21, 45–52 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Sorger, P. K., Dobles, M., Tournebize, R. & Hyman, A. A. Coupling cell division and cell death to microtubule dynamics. Curr. Opin. Cell Biol. 9, 807–814 (1997).

    Article  CAS  PubMed  Google Scholar 

  25. Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. & Draetta, G. Cyclin A is required at two points in the human cell cycle. EMBO J. 11, 961–971 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Rogakou, E. P., Nieves-Neira, W., Boon, C., Pommier, Y. & Bonner, W. M. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J. Biol. Chem. 275, 9390–9395 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Yeo, G. & Burge, C. B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Dewey, C. N., Rogozin, I. B. & Koonin, E. V. Compensatory relationship between splice sites and exonic splicing signals depending on the length of vertebrate introns. BMC Genom. 7, 311–319 (2006).

    Article  Google Scholar 

  30. Amit, M. et al. Differential GC content between exons and introns establishes distinct strategies of splice-site recognition. Cell Rep. 1, 543–556 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Hay, D. C., Sutherland, L., Clark, J. & Burdon, T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 22, 225–235 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Chang, Y. F., Imam, J. S. & Wilkinson, M. F. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51–74 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Ishigaki, Y., Li, X., Serin, G. & Maquat, L. E. Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Maquat, L. E. Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat. Rev. Mol. Cell Biol. 5, 89–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Chan, Y. S. et al. A PRC2-dependent repressive role of PRDM14 in human embryonic stem cells and induced pluripotent stem cell reprogramming. Stem Cells 31, 682–692 (2012).

    Article  Google Scholar 

  36. Fedor, M. J. Alternative splicing minireview series: combinatorial control facilitates splicing regulation of gene expression and enhances genome diversity. J. Biol. Chem. 283, 1209–1210 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Faustino, N. A. & Cooper, T. A. Pre-mRNA splicing and human disease. Genes Dev. 17, 419–437 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Yeo, G. W. et al. Alternative splicing events identified in human embryonic stem cells and neural progenitors. PLoS Comput. Biol. 3, 1951–1967 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Salomonis, N. et al. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors. PLoS Comput. Biol. 5, e1000553 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Salomonis, N. et al. Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation. Proc. Natl Acad. Sci. USA 107, 10514–10519 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yeo, G. W. et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gabut, M. et al. An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming. Cell 147, 132–146 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Han, H. et al. MBNL proteins repress ES-cell-specific alternative splicing and reprogramming. Nature 498, 241–245 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Le Cam, L., Lacroix, M., Ciemerych, M. A., Sardet, C. & Sicinski, P. The E4F protein is required for mitotic progression during embryonic cell cycles. Mol. Cell Biol. 24, 6467–6475 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. ENCODE Project Consortium, & Myers, R. M. et al. A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 9, e1001046 (2011).

    Article  Google Scholar 

  48. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  PubMed  Google Scholar 

  49. Paillusson, A., Hirschi, N., Vallan, C., Azzalin, C. M. & Muhlemann, O. A GFP-based reporter system to monitor nonsense-mediated mRNA decay. Nucleic Acids Res. 33, e54 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Wittmann, J., Hol, E. M. & Jack, H. M. hUPF2 silencing identifies physiologic substrates of mammalian nonsense-mediated mRNA decay. Mol. Cell. Biol. 26, 1272–1287 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Izquierdo, J. M., Alcalde, J., Carrascoso, I., Reyes, R. & Ludena, M. D. Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth. Biochem. J. 435, 337–344 (2011).

    Article  CAS  PubMed  Google Scholar 

  52. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the Agency for Science, Technology and Research (A*STAR) for their generous funding, and also for NIH R15 GM084407 to P.A.B. We acknowledge T. C. Peow, L-P. Yaw, T. Tng and G. K. Jee for their technical assistance. We acknowledge the Genome Technology Biology (GTB) group from the Genome Institute of Singapore (GIS) for their help and expertise in performing the RNA sequencing. We thank T-L. Huber for critical comments on the manuscript. J. Göke is supported by a fellowship within the Postdoc-Programme of the German Academic Exchange Service, DAAD.

Author information

Author notes

  1. Xinyi Lu and Jonathan Göke: These authors contributed equally to this work

Authors and Affiliations

  1. Gene Regulation Laboratory, Genome Institute of Singapore, 138672, Singapore

    Xinyi Lu, Jonathan Göke, Friedrich Sachs, Hongqing Liang & Huck-Hui Ng

  2. Department of Biological Sciences, National University of Singapore, 117543, Singapore

    Xinyi Lu & Huck-Hui Ng

  3. Department of Biochemistry, National University of Singapore, 117597, Singapore

    Friedrich Sachs & Huck-Hui Ng

  4. Department of Biology, Université de Sherbrooke, 2500 boulevard de l’Université Sherbrooke, Québec J1K 2R1, Canada

    Pierre-Étienne Jacques

  5. School of Biomedical Sciences, Lo Kwee Seong Integrated Biomedical Sciences Building, Chinese University of Hong Kong, Hong Kong 999077, China

    Bo Feng

  6. McGill University & Genome Quebec Innovation Center, 740 Dr Penfield Avenue, Montréal, Québec H3A 1A4, Canada

    Guillaume Bourque

  7. Department of Biological Sciences, Wright State University, Dayton, Ohio 45435, USA

    Paula A. Bubulya

  8. Graduate School for Integrative Sciences & Engineering, National University of Singapore, 117456, Singapore

    Huck-Hui Ng

  9. School of Biological Sciences, Nanyang Technological University, 637551, Singapore

    Huck-Hui Ng

Authors
  1. Xinyi Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathan Göke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Friedrich Sachs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pierre-Étienne Jacques
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongqing Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bo Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Guillaume Bourque
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paula A. Bubulya
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Huck-Hui Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.L.: conception, design, collection of data, data analysis and manuscript writing; J.G.: design, collection of data, data analysis and manuscript writing; F.S., H.L. and B.F.: collection and data analysis; P-É.J. and G.B.: collection and data analysis; P.A.B: design, and provision of study material; H-H.N.: conception, design, supervision, data interpretation and manuscript writing.

Corresponding author

Correspondence to Huck-Hui Ng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression pattern of SON in different cell types.

(a) Expression profile of SON according to probe 201086 and probe 214988 (Chip U133P2) of the AmaZonia human whole genome expression resource Supplementary Ref. S56. (b) SON expression changes during reprogramming, PRDM14 depletion and NFRKB depletion according to published microarray data5. OSKC: the 4 Yamanaka reprogramming factors: OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC(C). (c) SON expression in MRC5 cells and hESCs according to real-time PCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (d) SON expression during hESC differentiation induced by FGF removal. Mean±s.e.m. of biological triplicate data are shown in all graphs.

Supplementary Figure 2 Characterisation of the hESC differentiation phenotype after SON depletion.

(a) Western blot of SON protein in total cell extract from H1 hESCs. (b) Western blot of SON, pluripotency markers (OCT4 and NANOG) and differentiation markers (GATA6 and MSX1) after transfecting cells with constructs expressing control shRNA or SON shRNAs. β-ACTIN and GAPDH were used as loading control. (c) Immunostaining of pluripotency cell surface marker (SSEA4) and differentiation markers (GATA6 and MSX1) in hESCs. DAPI positively stains the nucleus. Scale bar, 20 μm. (d) Flow cytometry analysis of percentage of cells positive for pluripotency makers (OCT4, TRA-1-81 and TRA-1-60) and differentiation markers (GATA6 and MSX1) after SON knockdown. Isotype control is from secondary antibody staining alone. (e) Expression of housekeeping genes after SON depletion in hESCs according to real-time PCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (f) Number of TRA-1-60 positive colonies after reprogramming in the presence of SON shRNA. Mean±s.e.m. of biological triplicate data are shown in all graphs. OSKC: the 4 Yamanaka reprogramming factors: OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC(C). (g) Number of alkaline phosphatase colonies after SON depletion and rescue SON expression in hESCs. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 3 Influences of SON depletion on hESC survival and cell cycle progression.

(a) TUNEL assay for cell apoptosis after treating hESCs with control shRNA or SON shRNAs. Scale bar, 20 μm. (b) Expression of SON in MRC5 cells after SON knockdown for 4 days according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (c) Expression of SON at protein level after SON depletion in MRC5 cells according to Western blot. Mean±s.e.m. of biological triplicate data are shown in all graphs. (d) TUNEL assay after SON depletion in MRC5 cells. Scale bar, 20 μm. (e) Mitotic spindle staining after SON depletion. α-tubulin was immunostained for imaging. Representative examples of cells in mitosis are shown. After SON knockdown, cells with monopolar spindle pole or incomplete separation of bipolar spindle poles are shown. Scale bar, 2 μm. (f) Percentage of cells in different mitotic stages. α-Tubulin/Aurora A immunostaining was done in hESCs with or without SON knockdown. The number of mitotic cells in different phases and spindle pole status were counted. Monopolar and multipolar spindles are abnormal spindle structure. n = 100 cells. (g) Percentage of cells in mitosis calculated based on DAPI staining. Mean±s.e.m. of biological triplicate data are shown in all graphs. n>400 cells per experiment. (h) Morphology of hESCs after depleting SON and TUBA1C expression. Scale bar, 20 μm. shRNA1 was used for SON depletion. (i) TUNEL assay for cell apoptosis after treating cells with control shRNA or CYCLIN A2shRNAs. (j) Expression of pluripotency markers after TUBA1C depletion according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (k) CYCLIN A2depletion (right panel) according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs.

Supplementary Figure 4 Relationship between SON-knockdown induced differentiation and cell death.

(a) Time-course morphology of hESCs after depletion of SON. Scale bar, 20 μm. (b) Time-course expression of pluripotency markers after SON knockdown according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (c) Time-course expression of differentiation markers after SON knockdown according to qPCR. Mean±s.e.m. from biological triplicate data are shown. (d) Time-course analysis of cell cycle profile after SON depletion. Mean±s.e.m. from biological triplicate data are shown. (e) Cell cycle profile of hESCs after depletion of SON to various levels. Mean±s.e.m. from biological triplicate data are shown. (f) Flow cytometry analysis of percentage of cells positive for pluripotency makers (OCT4, TRA-1-81 and TRA-1-60) and differentiation markers (GATA6 and MSX1) after SON depletion in the presence of ROCK inhibitor. Isotype control is from secondary antibody staining alone. (g) Western blot of pluripotency marker OCT4 and differentiation marker GATA6 after SON depletion in the presence of ROCK inhibitor. (h) Flow cytometry analysis of TUNEL assay staining of cells treated with SON shRNA (shRNA1), TUBA1C shRNA or control shRNA and SON shRNA in the presence of ROCK inhibitor. (i) Flow cytometry analysis of pluripotency marker (SOX2) expression in γ-H2AX +/- populations. Isotype control is from secondary antibody staining alone. -, negatively stained population. +, positively stained population. (j) Flow cytometry analysis of pluripotency marker (TRA-1-60) expression by immunostaining in hESC population at different cell cycle stages after SON depletion. Isotype control is from secondary antibody staining alone. Cell cycle stages were determined by PI staining.

Supplementary Figure 5 Analysis and validation of RNA-seq results.

(a) Density plot showing the RNA-Seq data at the SON and ACTIN loci before and after SON depletion. RPKM values were calculated by normalising the number of reads in these loci by the length of these loci and the number of reads mapped to exons. (b) Gene ontology analysis of genes with decreased expression after SON depletion. (c) Histogram of the t-test statistic for intron inclusion events. The black line indicates the cutoff used in this study (-8) to select a stringent set of intron inclusion events. (d) Illustration to PCR primer locations in RT-PCR to detect inclusion of introns (left) or isoform switch (right) after SON depletion. (e) Analysis of intron inclusion events or isoform switches after SON depletion. Percentage of unspliced transcripts was measured according to band intensity. Control introns that are not regulated by SON are shown at the bottom of the figure. (f) Boxplot showing the normalised intron expression for SON-targeted introns as described in the main text. Intron expression was estimated by normalising the number of reads that mapped to the intron by intron length, exon read count and exon length. Significance was estimated using the Wilcoxon test.

Supplementary Figure 6 Characterisation of SON-targeted introns.

(a) Intron length for SON-targeted introns (estimated using intron expression) and unaffected introns from the same set of genes to account for gene to gene variation. (b) Intron length for SON-targeted introns (estimated using the ratio of spliced over unspliced reads) and non-targeted introns of SON. SONtargeted introns were identified by 3’ junction reads of the last exon only. (c) Splice strength for the 5’ splice site, estimated using MaxEntScan. (d) Splice strength for the 3’ splice site, estimated using MaxEntScan. (e) Nucleotide content for all introns in the genome (black), SON-targeted introns (red), and non-targeted introns from genes which had at least one affected intron (green). Left: 5’ end, right: 3’ end. (f) Nucleotide Frequencies for the 5’ and 3’ splice sites of introns unaffected introns from the same genes that had at least one intron targeted by SON to account for gene to gene variation (See Supplementary Fig. S6h). (g) Nucleotide Frequencies for the 5’ and 3’ splice sites of introns affected by SON knockdown, only genes with at least one affected and one unaffected intron were investigated to account for gene to gene variation (see Supplementary Fig. S6g). (h) Examples of noncandidate genes with weak splicing sites. Top panel: structure of genes Solid box: exon. Line: intron. Bottom panel: real-time PCR analysis of intron expression after SON depletion. Mean±s.e.m. from biological triplicate data are shown. (i) Morphology of hESCs after depletion of another weak splicing regulator TIA1. Scale bar, 20 μm. (j) Expression of pluripotency markers (left panel) and differentiation markers (right panel) after TIA1 depletion according to qPCR. Mean±s.e.m. of biological triplicate data are shown in all graphs. (k) Expression of SON regulated introns after depletion of TIA1 by qPCR. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 7 SON regulates the splicing of specific introns of pluripotency genes.

(a) Venn diagram showing the overlap of hESC specific genes (orange) with genes regulated by SON. The p-value was calculated using Fisher’s test. (b) Intron length of SON-targeted hESC specific introns. Significance was estimated using the Wilcoxon test. (c) Intron length of SON-targeted hESC-state-maintaining genes. Significance was estimated using the Wilcoxon test. (d) Intron length of SON-targeted cell survival genes. Significance was estimated using the Wilcoxon test. (e) Time-course expression fold change of candidate introns after SON depletion. The fold changes of intron expression levels were normalised to gene expression fold change (measured by primers designed for constitutive exonic sequence) fold change (set as 1). TUBA1B intron 2 as negative control. Mean±s.e.m. from biological triplicate data are shown. (f) Expression of introns and exons in the OCT4 gene after SON depletion. Solid box: exon. Line: introns. Arrows: locations of primers. Mean±s.e.m. from biological triplicate data are shown. (g) Gene structure of NANOG and position of primers and qPCR analysis of expression change of NANOG introns after SON depletion for 3 days. Solid box: exon.Line: introns. Arrows: locations of primer pairs. SJ: splice junction. Mean±s.e.m. from biological triplicate data are shown.

Supplementary Figure 8 Analysis of SON regulated alternative splicing.

(a) Venn diagram showing the overlap of pluripotency genes (green) with genes that show alternative splicing after depletion of SON. The p-value was calculated using Fisher’s test. (b) Venn diagram showing the overlap of self-renewal genes (red) with genes that show alternative splicing after depletion of SON. Genes essential for hESC survival were defined as genes with average cell survival Nscore<−2 (Supplementary Table S2 from genome-wide RNAi screening in hESCs5). The P-value was calculated using Fisher’s test.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2659 kb)

Supplementary Table 1

Supplementary Information (XLSX 319 kb)

Supplementary Table 2

Supplementary Information (XLSX 113 kb)

Supplementary Table 3

Supplementary Information (XLSX 15 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, X., Göke, J., Sachs, F. et al. SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells. Nat Cell Biol 15, 1141–1152 (2013). https://doi.org/10.1038/ncb2839

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2839

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing