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.

A complex network of factors with overlapping affinities represses splicing through intronic elements

Subjects

Abstract

To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from introns. Motifs were validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs according to their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, to recover 24 known and new splicing regulators. Specific domains in selected factors were sufficient to confer intronic-splicing-silencer activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, which revealed a complex overlapping network of protein-RNA interactions. This arrangement enables individual cis elements to function differently in distinct cellular contexts, depending on the spectrum of regulatory factors present.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Fluorescence-activated screen for intronic splicing silencers (FAS-ISS) identifies 102 unique ISS 10-mers.
Figure 2: Identification of core ISS motifs and exemplars and validation of activity.
Figure 3: ISS motifs regulate splicing from an exonic context.
Figure 4: Classification of ISSs on the basis of genomic distribution yields clusters with similar context-dependent activity.
Figure 5: Identification and validation of ISS-associated splicing repressors.
Figure 6: ISSs are recognized by a complex overlapping network of factors.

Accession codes

Accessions

European Nucleotide Archive

References

  1. Wang, Z. & Burge, C.B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813 (2008).

    Article  CAS  Google Scholar 

  2. Matlin, A.J., Clark, F. & Smith, C.W. Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398 (2005).

    Article  CAS  Google Scholar 

  3. Blencowe, B.J. Alternative splicing: new insights from global analyses. Cell 126, 37–47 (2006).

    Article  CAS  Google Scholar 

  4. Liu, H.X., Zhang, M. & Krainer, A.R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12, 1998–2012 (1998).

    Article  CAS  Google Scholar 

  5. Fairbrother, W.G., Yeh, R.F., Sharp, P.A. & Burge, C.B. Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007–1013 (2002).

    Article  CAS  Google Scholar 

  6. Zhang, X.H. & Chasin, L.A. Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev. 18, 1241–1250 (2004).

    Article  CAS  Google Scholar 

  7. Wang, Z. et al. Systematic identification and analysis of exonic splicing silencers. Cell 119, 831–845 (2004).

    Article  CAS  Google Scholar 

  8. Goren, A. et al. Comparative analysis identifies exonic splicing regulatory sequences–the complex definition of enhancers and silencers. Mol. Cell 22, 769–781 (2006).

    Article  CAS  Google Scholar 

  9. Yu, Y. et al. Dynamic regulation of alternative splicing by silencers that modulate 5′ splice site competition. Cell 135, 1224–1236 (2008).

    Article  CAS  Google Scholar 

  10. Culler, S.J., Hoff, K.G., Voelker, R.B., Berglund, J.A. & Smolke, C.D. Functional selection and systematic analysis of intronic splicing elements identifies active sequence motifs and associated splicing factors. Nucleic Acids Res. 38, 5152–5165 (2010).

    Article  CAS  Google Scholar 

  11. Sharma, S., Kohlstaedt, L.A., Damianov, A., Rio, D.C. & Black, D.L. Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. Nat. Struct. Mol. Biol. 15, 183–191 (2008).

    Article  CAS  Google Scholar 

  12. Kashima, T., Rao, N. & Manley, J.L. An intronic element contributes to splicing repression in spinal muscular atrophy. Proc. Natl. Acad. Sci. USA 104, 3426–3431 (2007).

    Article  CAS  Google Scholar 

  13. Hui, J. et al. Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing. EMBO J. 24, 1988–1998 (2005).

    Article  CAS  Google Scholar 

  14. Blanchette, M. & Chabot, B. Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization. EMBO J. 18, 1939–1952 (1999).

    Article  CAS  Google Scholar 

  15. Kanopka, A., Muhlemann, O. & Akusjarvi, G. Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA. Nature 381, 535–538 (1996).

    Article  CAS  Google Scholar 

  16. Ibrahim, E.C., Schaal, T.D., Hertel, K.J., Reed, R. & Maniatis, T. Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers. Proc. Natl. Acad. Sci. USA 102, 5002–5007 (2005).

    Article  CAS  Google Scholar 

  17. Shen, M. & Mattox, W. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position. Nucleic Acids Res. 40, 428–437 (2012).

    Article  CAS  Google Scholar 

  18. McNally, L.M. & McNally, M.T. SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element. J. Virol. 70, 1163–1172 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Del Gatto-Konczak, F., Olive, M., Gesnel, M.C. & Breathnach, R. hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell Biol. 19, 251–260 (1999).

    Article  CAS  Google Scholar 

  20. Hua, Y., Vickers, T.A., Okunola, H.L., Bennett, C.F. & Krainer, A.R. Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am. J. Hum. Genet. 82, 834–848 (2008).

    Article  CAS  Google Scholar 

  21. Tange, T.O., Damgaard, C.K., Guth, S., Valcarcel, J. & Kjems, J. The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element. EMBO J. 20, 5748–5758 (2001).

    Article  CAS  Google Scholar 

  22. Wang, Y., Ma, M., Xiao, X. & Wang, Z. Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules. Nat. Struct. Mol. Biol. 19, 1044–1052 (2012).

    Article  CAS  Google Scholar 

  23. Wang, Z., Xiao, X., Van Nostrand, E. & Burge, C.B. General and specific functions of exonic splicing silencers in splicing control. Mol. Cell 23, 61–70 (2006).

    Article  CAS  Google Scholar 

  24. Lim, K.H., Ferraris, L., Filloux, M.E., Raphael, B.J. & Fairbrother, W.G. Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes. Proc. Natl. Acad. Sci. USA 108, 11093–11098 (2011).

    Article  CAS  Google Scholar 

  25. Huang, C. et al. A structured RNA in HBV PRE represses alternative splicing in a sequence-independent and position-dependent manner. FEBS J. 278, 1533–1546 (2011).

    Article  CAS  Google Scholar 

  26. Pervouchine, D.D. et al. Evidence for widespread association of mammalian splicing and conserved long-range RNA structures. RNA 18, 1–15 (2012).

    Article  CAS  Google Scholar 

  27. Dominski, Z., Yang, X.C., Kaygun, H., Dadlez, M. & Marzluff, W.F. A 3′ exonuclease that specifically interacts with the 3′ end of histone mRNA. Mol. Cell 12, 295–305 (2003).

    Article  CAS  Google Scholar 

  28. Rothrock, C.R., House, A.E. & Lynch, K.W. HnRNP L represses exon splicing via a regulated exonic splicing silencer. EMBO J. 24, 2792–2802 (2005).

    Article  CAS  Google Scholar 

  29. Nielsen, F.C., Nielsen, J. & Christiansen, J. A family of IGF-II mRNA binding proteins (IMP) involved in RNA trafficking. Scand. J. Clin. Lab. Invest. Suppl. 234, 93–99 (2001).

    Article  CAS  Google Scholar 

  30. Allemand, E., Hastings, M.L., Murray, M.V., Myers, M.P. & Krainer, A.R. Alternative splicing regulation by interaction of phosphatase PP2Cgamma with nucleic acid-binding protein YB-1. Nat. Struct. Mol. Biol. 14, 630–638 (2007).

    Article  CAS  Google Scholar 

  31. Krainer, A.R., Conway, G.C. & Kozak, D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 4, 1158–1171 (1990).

    Article  CAS  Google Scholar 

  32. Muta, T., Kang, D., Kitajima, S., Fujiwara, T. & Hamasaki, N. p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation. J. Biol. Chem. 272, 24363–24370 (1997).

    Article  CAS  Google Scholar 

  33. Oberstrass, F.C. et al. Structure of PTB bound to RNA: specific binding and implications for splicing regulation. Science 309, 2054–2057 (2005).

    Article  CAS  Google Scholar 

  34. Spellman, R., Llorian, M. & Smith, C.W. Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB and ROD1. Mol. Cell 27, 420–434 (2007).

    Article  CAS  Google Scholar 

  35. Jin, W., Bruno, I.G., Xie, T.X., Sanger, L.J. & Cote, G.J. Polypyrimidine tract-binding protein down-regulates fibroblast growth factor receptor 1 α-exon inclusion. Cancer Res. 63, 6154–6157 (2003).

    CAS  PubMed  Google Scholar 

  36. Côté, J., Dupuis, S. & Wu, J.Y. Polypyrimidine track-binding protein binding downstream of caspase-2 alternative exon 9 represses its inclusion. J. Biol. Chem. 276, 8535–8543 (2001).

    Article  Google Scholar 

  37. Das, R. et al. SR proteins function in coupling RNAP II transcription to pre-mRNA splicing. Mol. Cell 26, 867–881 (2007).

    Article  CAS  Google Scholar 

  38. Chou, M.Y., Rooke, N., Turck, C.W. & Black, D.L. hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Mol. Cell Biol. 19, 69–77 (1999).

    Article  CAS  Google Scholar 

  39. Kashima, T., Rao, N., David, C.J. & Manley, J.L. hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing. Hum. Mol. Genet. 16, 3149–3159 (2007).

    Article  CAS  Google Scholar 

  40. Hui, J., Stangl, K., Lane, W.S. & Bindereif, A. HnRNP L stimulates splicing of the eNOS gene by binding to variable-length CA repeats. Nat. Struct. Biol. 10, 33–37 (2003).

    Article  CAS  Google Scholar 

  41. Chen, M. & Manley, J.L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat. Rev. Mol. Cell Biol. 10, 741–754 (2009).

    Article  CAS  Google Scholar 

  42. Graveley, B.R. & Maniatis, T. Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing. Mol. Cell 1, 765–771 (1998).

    Article  CAS  Google Scholar 

  43. Shen, H., Kan, J.L. & Green, M.R. Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly. Mol. Cell 13, 367–376 (2004).

    Article  CAS  Google Scholar 

  44. Graveley, B.R., Hertel, K.J. & Maniatis, T. A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers. EMBO J. 17, 6747–6756 (1998).

    Article  CAS  Google Scholar 

  45. Wang, Y., Cheong, C.G., Hall, T.M. & Wang, Z. Engineering splicing factors with designed specificities. Nat. Methods 6, 825–830 (2009).

    Article  CAS  Google Scholar 

  46. Cheong, C.G. & Hall, T.M. Engineering RNA sequence specificity of Pumilio repeats. Proc. Natl. Acad. Sci. USA 103, 13635–13639 (2006).

    Article  CAS  Google Scholar 

  47. Tourrière, H. et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 160, 823–831 (2003).

    Article  Google Scholar 

  48. Ufer, C. et al. Translational regulation of glutathione peroxidase 4 expression through guanine-rich sequence-binding factor 1 is essential for embryonic brain development. Genes Dev. 22, 1838–1850 (2008).

    Article  CAS  Google Scholar 

  49. Michlewski, G., Guil, S., Semple, C.A. & Caceres, J.F. Posttranscriptional regulation of miRNAs harboring conserved terminal loops. Mol. Cell 32, 383–393 (2008).

    Article  CAS  Google Scholar 

  50. Guil, S., Long, J.C. & Caceres, J.F. hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol. Cell Biol. 26, 5744–5758 (2006).

    Article  CAS  Google Scholar 

  51. Boutz, P.L. et al. A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons. Genes Dev. 21, 1636–1652 (2007).

    Article  CAS  Google Scholar 

  52. Caputi, M. & Zahler, A.M. Determination of the RNA binding specificity of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/H'/F/2H9 family. J. Biol. Chem. 276, 43850–43859 (2001).

    Article  CAS  Google Scholar 

  53. Schaub, M.C., Lopez, S.R. & Caputi, M. Members of the heterogeneous nuclear ribonucleoprotein H family activate splicing of an HIV-1 splicing substrate by promoting formation of ATP-dependent spliceosomal complexes. J. Biol. Chem. 282, 13617–13626 (2007).

    Article  CAS  Google Scholar 

  54. Chen, C.D., Kobayashi, R. & Helfman, D.M. Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev. 13, 593–606 (1999).

    Article  CAS  Google Scholar 

  55. Xiao, X., Wang, Z., Jang, M. & Burge, C.B. Coevolutionary networks of splicing cis-regulatory elements. Proc. Natl. Acad. Sci. USA 104, 18583–18588 (2007).

    Article  CAS  Google Scholar 

  56. Xiao, X. et al. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat. Struct. Mol. Biol. 16, 1094–1100 (2009).

    Article  CAS  Google Scholar 

  57. Katz, Y., Wang, E.T., Airoldi, E.M. & Burge, C.B. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods 7, 1009–1015 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Hui (Shanghai Institute of Biological Science, Shanghai, China) and A. Willis (University of Nottingham, UK) for providing expression constructs of trans factors and B. Graveley (University of Connecticut Health Center, Farmington, Connecticut, USA) for constructs containing RS domains. We thank T. Nilsen and A. Berglund for critical reading of manuscripts and Z. Dominski and B. Marzluff for helping in RNA affinity purifications. This work was supported by an American Heart Association grant (0865329E) and US National Institutes of Health grant (R01CA158283) to Z.W. and (2-R01-GM085319) to C.B.B.

Author information

Authors and Affiliations

Authors

Contributions

Y.W., C.B.B. and Z.W. designed the research. Y.W., Z.W., J.Z., K.L. and R.C. performed the experiments. M.M., X.X. and A.R. developed computational methods to analyze the data. Y.W., C.B.B. and Z.W. wrote the paper.

Corresponding authors

Correspondence to Christopher B Burge or Zefeng Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of FAS-ISS decamers.

(a) Flow cytometry profile of 293 FlipIn cells transfected with the pZW11 reporter inserted with random library. After selection for stable integration, all hygromycin resistant cells were pooled and analyzed by flowcytometry. Both red and green fluorescence signals were measured to correct for self-fluorescence background. The GFP-positive cells (R1 region) were sorted using a Cytomation MoFlo high-speed sorter into 96 well plates to recover all of the ISS sequences. (b) Validation of FAS-ISS decamer activity. To validate the silencer activities of the newly identified ISS decamers, 293T cells were transiently transfected with pZW11 containing 16 arbitrarily selected ISS decamers and control sequence. The GFP-positive cells were examined by flow cytometry at 24h after transfection. (c) The frequencies of mononucleotide in the screened ISS decamer set. (d) The frequencies and odds ratios of dinucleotides in the screened ISS decamers set.

Supplementary Figure 2 Diverse functions of ISSs when inserted in the exons or between two 5′ SS.

(a) The ISS exemplars of each group were inserted into the exon of a splicing reporter and transiently transfected into both HeLa and 293T cells. After 48 hours, RNAs were isolated from the transfected cells to determine the functions of ISSs by RT-PCR. (b) The representative ISSs of each group were inserted between two 5′ SS in the exonic extension region of the mini-gene reporter. The resulting reporters were transiently transfected into HeLa and 293T cells. After 48 hours, RNAs were extracted from the transfected cells and the functions of ISSs were examined by RT-PCR and shown in the gel figures.

Supplementary Figure 3 The positional frequency of FAS-ISS k-mers in different premRNA locations.

Distribution of ISS k-mers near the constitutive exon (black) and skipped exon (red). The number of transcripts containing ISS k-mers divided by total number of transcripts at each position is plotted as ISS frequency. The first and last 50 bases of exons and the first and last ~200 bases of introns are shown, excluding the last 20 bases of the upstream intron and the first 10 bases of the downstream intron to avoid overlaps with the splice site motifs.

Supplementary Figure 4 Depletion or overexpression of hnRNP L and YB1 proteins.

(a) 293T cells were transfected with the siRNA of HNRNP L and YB1. After 48 hours of siRNA transfection, we transfected 0.2 μg splicing mini-gene reporters containing ISS group U or group D into the cells respectively, and harvested the cells 24 hours after the second transfection to examine the protein levels of HNRNP L and YB1 through Western blot, and the tubulin level was detected as loading control. GRSF1 siRNA was used as the specificity control. The asterisk indicated a non-specific protein that cross-react with HNRNP L antibody. (b) 293T cells were transiently transfected with expression vectors of HNRNP L and YB1. After 72 hours, proteins were extracted from the transfected cells to determine the expression levels of HNRNP L and YB1 with western blot. The asterisk indicated a non-specific protein that cross-react with HNRNP L antibody. The tubulin level was examined as loading control. (c) 293T cells were transfected with siRNA of scramble control or HNRNP L. After 48 hours of siRNA transfection, the cells were cotransfected with 0.2 μg splicing reporter containing ISS group U or group D and FLAGYB1 expression vector (lane 3 and 6). The cells were harvested after another 24 hours for the protein analysis. The tubulin level was measured as loading control.

Supplementary Figure 5 The SDS-PAGE gel of putative protein factors that bind to different ISS groups.

Biotinylated RNA oligos of each ISS group were incubated with HeLa whole cell extract, bound to streptavidin beads and washed, the RNA-protein complex were eluted and separated on a SDS-PAGE gel. The specific bands (marked with a dot and labeled according to each group) were cut and identified by mass spectrometry. Two batches of the affinity purification experiments were carried out for group D ISS, and we separated two samples in the same gel for protein identification.

Supplementary Figure 6 Purification of recombinant proteins for measurement of direct RNA-protein binding.

The putative trans-factors binding to groups F, H, I were cloned into bacterial expression system (pT7HtB ) and purified with His GraviTrap Kit. The final protein products were assayed with SDS-PAGE to check purity, and were later used in Biacore assay to measure the direct RNA-protein binding. Three fractions eluded from Ni column were shown for hnRNP A0, A2, A1 and D, and the elution fractions were combined for hnRNP DL

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–7 and Supplementary Note (PDF 10417 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Xiao, X., Zhang, J. et al. A complex network of factors with overlapping affinities represses splicing through intronic elements. Nat Struct Mol Biol 20, 36–45 (2013). https://doi.org/10.1038/nsmb.2459

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2459

This article is cited by

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