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:

Control of alternative splicing through siRNA-mediated transcriptional gene silencing

Nature Structural & Molecular Biology volume 16, pages 717–724 (2009)Cite this article

Abstract

When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1α and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.

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: Intronic siRNAs affect fibronectin alternative splicing in Hep3B cells.
Figure 2: Effects of intronic siRNAs on EDI alternative splicing of the endogenous FN1 gene in HeLa cells.
Figure 3: Factors influencing the siRNA effect on EDI alternative splicing.
Figure 4: Importance of the chromatin state in the TGS-AS pathway.
Figure 5: I33as transfection promotes histone methylation at the gene target region.
Figure 6: Depletion of AGO1 and DCR affects many ASEs.
Figure 7: Model for TGS-AS.

Similar content being viewed by others

References

  1. Kornblihtt, A.R. Chromatin, transcript elongation and alternative splicing. Nat. Struct. Mol. Biol. 13, 5–7 (2006).

    Article  CAS  Google Scholar 

  2. Lorincz, M.C., Dickerson, D.R., Schmitt, M. & Groudine, M. Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat. Struct. Mol. Biol. 11, 1068–1075 (2004).

    Article  CAS  Google Scholar 

  3. Batsché, E., Yaniv, M. & Muchardt, C. The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat. Struct. Mol. Biol. 13, 22–29 (2006).

    Article  Google Scholar 

  4. Schor, I.E., Rascovan, N., Pelisch, F., Allo, M. & Kornblihtt, A.R. Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing. Proc. Natl. Acad. Sci. USA 106, 4325–4330 (2009).

    Article  CAS  Google Scholar 

  5. Kim, D.H., Villeneuve, L.M., Morris, K.V. & Rossi, J.J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 13, 793–797 (2006).

    Article  CAS  Google Scholar 

  6. Morris, K.V., Chan, S.W., Jacobsen, S.E. & Looney, D.J. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305, 1289–1292 (2004).

    Article  CAS  Google Scholar 

  7. Suzuki, K. et al. Closed chromatin architecture is induced by an RNA duplex targeting the HIV-1 promoter region. J. Biol. Chem. 283, 23353–23363 (2008).

    Article  CAS  Google Scholar 

  8. Kuramochi-Miyagawa, S. et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev. 22, 908–917 (2008).

    Article  CAS  Google Scholar 

  9. González, S., Pisano, D.G. & Serrano, M. Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs. Cell Cycle 7, 2601–2608 (2008).

    Article  Google Scholar 

  10. Zhang, M.X. et al. Effect of 27nt small RNA on endothelial nitric-oxide synthase expression. Mol. Biol. Cell 19, 3997–4005 (2008).

    Article  CAS  Google Scholar 

  11. Zhang, M.X. et al. Biogenesis of short intronic repeat 27-nucleotide small RNA from endothelial nitric-oxide synthase gene. J. Biol. Chem. 283, 14685–14693 (2008).

    Article  CAS  Google Scholar 

  12. Tay, Y., Zhang, J., Thomson, A.M., Lim, B. & Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature (2008).

  13. Kim, D.H., Saetrom, P., Snove, O. Jr. & Rossi, J.J. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc. Natl. Acad. Sci. USA 105, 16230–16235 (2008).

    Article  CAS  Google Scholar 

  14. de la Mata, M. et al. A slow RNA polymerase II affects alternative splicing in vivo. Mol. Cell 12, 525–532 (2003).

    Article  CAS  Google Scholar 

  15. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  16. Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).

    Article  CAS  Google Scholar 

  17. Persengiev, S.P., Zhu, X. & Green, M.R. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18 (2004).

    Article  CAS  Google Scholar 

  18. Weinberg, M.S. et al. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12, 256–262 (2006).

    Article  CAS  Google Scholar 

  19. Han, J., Kim, D. & Morris, K.V. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc. Natl. Acad. Sci. USA 104, 12422–12427 (2007).

    Article  CAS  Google Scholar 

  20. Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    Article  CAS  Google Scholar 

  21. Janowski, B.A. et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat. Struct. Mol. Biol. 13, 787–792 (2006).

    Article  CAS  Google Scholar 

  22. de la Mata, M. & Kornblihtt, A.R. RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20. Nat. Struct. Mol. Biol. 13, 973–980 (2006).

    Article  CAS  Google Scholar 

  23. Tong, W., Kulaeva, O.I., Clark, D.J. & Lutter, L.C. Topological analysis of plasmid chromatin from yeast and mammalian cells. J. Mol. Biol. 361, 813–822 (2006).

    Article  CAS  Google Scholar 

  24. Wang, G., Liu, T., Wei, L.N., Law, P.Y. & Loh, H.H. DNA methylation-related chromatin modification in the regulation of mouse delta-opioid receptor gene. Mol. Pharmacol. 67, 2032–2039 (2005).

    Article  CAS  Google Scholar 

  25. Yokoyama, R., Pannuti, A., Ling, H., Smith, E.R. & Lucchesi, J.C. A plasmid model system shows that Drosophila dosage compensation depends on the global acetylation of histone H4 at lysine 16 and is not affected by depletion of common transcription elongation chromatin marks. Mol. Cell. Biol. 27, 7865–7870 (2007).

    Article  CAS  Google Scholar 

  26. Blau, J. et al. Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16, 2044–2055 (1996).

    Article  CAS  Google Scholar 

  27. Kubicek, S. et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol. Cell 25, 473–481 (2007).

    Article  CAS  Google Scholar 

  28. Cam, H.P., Chen, E.S. & Grewal, S.I. Transcriptional scaffolds for heterochromatin assembly. Cell 136, 610–614 (2009).

    Article  CAS  Google Scholar 

  29. Moazed, D. Small RNAs in transcriptional gene silencing and genome defence. Nature 457, 413–420 (2009).

    Article  CAS  Google Scholar 

  30. Umlauf, D., Goto, Y. & Feil, R. Site-specific analysis of histone methylation and acetylation. Methods Mol. Biol. 287, 99–120 (2004).

    CAS  PubMed  Google Scholar 

  31. Kapranov, P. et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316, 1484–1488 (2007).

    Article  CAS  Google Scholar 

  32. Klinck, R. et al. Multiple alternative splicing markers for ovarian cancer. Cancer Res. 68, 657–663 (2008).

    Article  CAS  Google Scholar 

  33. Kim, V.N., Han, J. & Siomi, M.C. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139 (2009).

    Article  CAS  Google Scholar 

  34. Okamura, K. & Lai, E.C. Endogenous small interfering RNAs in animals. Nat. Rev. Mol. Cell Biol. 9, 673–678 (2008).

    Article  CAS  Google Scholar 

  35. Tam, O.H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).

    Article  CAS  Google Scholar 

  36. Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).

    Article  CAS  Google Scholar 

  37. Yang, N. & Kazazian, H.H. Jr. L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells. Nat. Struct. Mol. Biol. 13, 763–771 (2006).

    Article  CAS  Google Scholar 

  38. Schwartz, J.C. et al. Antisense transcripts are targets for activating small RNAs. Nat. Struct. Mol. Biol. 15, 842–848 (2008).

    Article  CAS  Google Scholar 

  39. Morris, K.V., Santoso, S., Turner, A.M., Pastori, C. & Hawkins, P.G. Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet. 4, e1000258 (2008).

    Article  Google Scholar 

  40. Haussecker, D. & Proudfoot, N.J. Dicer-dependent turnover of intergenic transcripts from the human beta-globin gene cluster. Mol. Cell. Biol. 25, 9724–9733 (2005).

    Article  CAS  Google Scholar 

  41. Tufarelli, C. et al. Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat. Genet. 34, 157–165 (2003).

    Article  CAS  Google Scholar 

  42. Yu, W. et al. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature 451, 202–206 (2008).

    Article  CAS  Google Scholar 

  43. Rinn, J.L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007).

    Article  CAS  Google Scholar 

  44. Kadener, S. et al. Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing. EMBO J. 20, 5759–5768 (2001).

    Article  CAS  Google Scholar 

  45. Nogués, G., Kadener, S., Cramer, P., Bentley, D. & Kornblihtt, A.R. Transcriptional activators differ in their abilities to control alternative splicing. J. Biol. Chem. 277, 43110–43114 (2002).

    Article  Google Scholar 

  46. Cramer, P. et al. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol. Cell 4, 251–258 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Bertucci, M. Blaustein, F. Pelisch, M. Muñoz, A. Srebrow, G. Risso, L.G. Acuña, M.G. Herz, N. Tilgner, I. Listerman, M. Bühler, J. Martínez, K. Neugebauer and K. Morris for their support and useful discussions; and Boehringer Ingelheim Pharmaceuticals (Biomolecular Screening, Department of Medicinal Chemistry) for the gift of BIX-01294. This work was supported by grants to A.R.K. from the Fundación Antorchas, the Agencia Nacional de Promoción de Ciencia y Tecnología of Argentina, the University of Buenos Aires and the European Alternative Splicing Network. S.A.E., R.K. and B.C. acknowledge support from Genome Canada and Genome Quebec. M.A. is the recipient of a fellowship and A.R.K. is a career investigator from the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina. B.C. holds a Canada Research Chair in functional genomics. A.R.K. is an international research scholar of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Departamento de Fisiología, Laboratorio de Fisiología y Biología Molecular, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina

    Mariano Alló, Valeria Buggiano, Juan P Fededa, Ezequiel Petrillo, Ignacio Schor, Manuel de la Mata & Alberto R Kornblihtt

  2. ICREA and Universitat Pompeu Fabra, Barcelona, Spain

    Eneritz Agirre, Mireya Plass & Eduardo Eyras

  3. Laboratoire de Génomique Fonctionnelle, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Québec, Canada

    Sherif Abou Elela, Roscoe Klinck & Benoit Chabot

Authors
  1. Mariano Alló
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Valeria Buggiano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juan P Fededa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ezequiel Petrillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ignacio Schor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manuel de la Mata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eneritz Agirre
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mireya Plass
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eduardo Eyras
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sherif Abou Elela
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Roscoe Klinck
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Benoit Chabot
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Alberto R Kornblihtt
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.A. proposed, designed and conducted most of the experiments and prepared the manuscript; V.B., J.P.F., E.P., I.S. and M.d.l.M. conducted some experiments; E.A., M.P. and E.E. conducted the bioinformatics analysis; S.A.E., R.K. and B.C. conducted the PCR panel experiment; and A.R.K. coordinated the work and prepared the manuscript.

Corresponding author

Correspondence to Alberto R Kornblihtt.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 568 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alló, M., Buggiano, V., Fededa, J. et al. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat Struct Mol Biol 16, 717–724 (2009). https://doi.org/10.1038/nsmb.1620

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1620

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