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:

RNA splicing promotes translation and RNA surveillance

Nature Structural & Molecular Biology volume 12, pages 801–809 (2005)Cite this article

Abstract

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.

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 VDJ exon is dispensable for strong downregulation of mRNAs that contain a PTC.
Figure 2: Mutations in the IVS2 3′ splice site reduce the downregulation of TCRβ transcripts that contain a PTC.
Figure 3: A mutation in the IVS4 3′ splice site reduces the downregulation of TCRβ transcripts that contain a PTC.
Figure 4: PTC position dictates whether efficient splicing promotes mRNA downregulation.
Figure 5: Splicing efficiency dictates the magnitude of downregulation of TPI mRNAs that contain a PTC.
Figure 6: The chimeric intron is not essential for strong downregulation of TPI minigene mRNAs that contain a PTC.
Figure 7: Strengthening splicing signals elicits strong downregulation by an upstream PTC, not a downstream PTC.
Figure 8: Reducing splicing efficiency markedly reduces protein levels.

Similar content being viewed by others

References

  1. Frischmeyer, P.A. & Dietz, H.C. Nonsense-mediated mRNA decay in health and disease. Hum. Mol. Genet. 8, 1893–1900 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Jacobson, A. & Peltz, S.W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu. Rev. Biochem. 65, 693–739 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. 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 

  4. Wilkinson, M.F. A new function for nonsense-mediated mRNA-decay factors. Trends Genet. 21, 143–148 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. & Kulozik, A.E. Nonsense-mediated decay approaches the clinic. Nat. Genet. 36, 801–808 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Carter, M.S., Li, S. & Wilkinson, M.F. A splicing-dependent regulatory mechanism that detects translation signals. EMBO J. 15, 5965–5975 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brocke, K.S., Neu-Yilik, G., Gehring, N.H., Hentze, M.W. & Kulozik, A.E. The human intronless melanocortin 4-receptor gene is NMD insensitive. Hum. Mol. Genet. 11, 331–335 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Maquat, L.E. & Li, X. Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay. RNA 7, 445–456 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Le Hir, H., Izaurralde, E., Maquat, L.E. & Moore, M.J. The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19, 6860–6869 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Wagner, E. & Lykke-Andersen, J. mRNA surveillance: the perfect persist. J. Cell Sci. 115, 3033–3038 (2002).

    CAS  PubMed  Google Scholar 

  11. Li, S. & Wilkinson, M.F. Nonsense surveillance in lymphocytes? Immunity 8, 135–141 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Gudikote, J.P. & Wilkinson, M.F. T-cell receptor sequences that elicit strong down-regulation of premature termination codon-bearing transcripts. EMBO J. 21, 125–134 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cheng, J., Fogel-Petrovic, M. & Maquat, L.E. Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol. Cell. Biol. 10, 5215–5225 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Carter, M.S. et al. A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro. J. Biol. Chem. 270, 28995–29003 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. 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  PubMed  PubMed Central  Google Scholar 

  16. Audibert, A., Weil, D. & Dautry, F. In vivo kinetics of mRNA splicing and transport in mammalian cells. Mol. Cell. Biol. 22, 6706–6718 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, S., Leonard, D. & Wilkinson, M.F. T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism. J. Exp. Med. 185, 985–992 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang, J., Vock, V.M., Li, S., Olivas, O.R. & Wilkinson, M.F. A quality control pathway that down-regulates aberrant T-cell receptor (TCR) transcripts by a mechanism requiring UPF2 and translation. J. Biol. Chem. 277, 18489–18493 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Nott, A., Le Hir, H. & Moore, M.J. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes Dev. 18, 210–222 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Buzina, A. & Shulman, M.J. Infrequent translation of a nonsense codon is sufficient to decrease mRNA level. Mol. Biol. Cell 10, 515–524 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Buhler, M., Paillusson, A. & Muhlemann, O. Efficient downregulation of immunoglobulin mu mRNA with premature translation-termination codons requires the 5′-half of the VDJ exon. Nucleic Acids Res. 32, 3304–3315 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Kessler, O. & Chasin, L.A. Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA. Mol. Cell. Biol. 16, 4426–4435 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Urlaub, G., Mitchell, P.J., Ciudad, C.J. & Chasin, L.A. Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol. Cell. Biol. 9, 2868–2880 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lewis, B.P., Green, R.E. & Brenner, S.E. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad. Sci. USA 100, 189–192 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Mendell, J.T., Sharifi, N.A., Meyers, J.L., Martinez-Murillo, F. & Dietz, H.C. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat. Genet. 36, 1073–1078 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Mitrovich, Q.M. & Anderson, P. Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev. 14, 2173–2184 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Morrison, M., Harris, K.S. & Roth, M.B. smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 94, 9782–9785 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wiegand, H.L., Lu, S. & Cullen, B.R. Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proc. Natl. Acad. Sci. USA 100, 11327–11332 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Custodio, N. et al. In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei. RNA 10, 622–633 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sanford, J.R., Gray, N.K., Beckmann, K. & Caceres, J.F. A novel role for shuttling SR proteins in mRNA translation. Genes Dev. 18, 755–768 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, Z. & Krainer, A.R. Involvement of SR Proteins in mRNA Surveillance. Mol. Cell 16, 597–607 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Zolotukhin, A.S., Tan, W., Bear, J., Smulevitch, S. & Felber, B.K. U2AF participates in the binding of TAP (NXF1) to mRNA. J. Biol. Chem. 277, 3935–3942 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Dahlberg, J.E., Lund, E. & Goodwin, E.B. Nuclear translation: what is the evidence? RNA 9, 1–8 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Iborra, F.J., Jackson, D.A. & Cook, P.R. The case for nuclear translation. J. Cell Sci. 117, 5713–5720 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Wilkinson, M.F. & Shyu, A.B. RNA surveillance by nuclear scanning? Nat. Cell Biol. 4, E144–E147 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Aoufouchi, S., Yelamos, J. & Milstein, C. Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis. Cell 85, 415–422 (1996).

    Article  CAS  PubMed  Google Scholar 

  37. Gersappe, A., Burger, L. & Pintel, D.J. A premature termination codon in either exon of minute virus of mice P4 promoter-generated pre-mRNA can inhibit nuclear splicing of the intervening intron in an open reading frame-dependent manner. J. Biol. Chem. 274, 22452–22458 (1999).

    Article  CAS  PubMed  Google Scholar 

  38. Lozano, F., Maertzdorf, B., Pannell, R. & Milstein, C. Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing. EMBO J. 13, 4617–4622 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Muhlemann, O. et al. Precursor RNAs harboring nonsense codons accumulate near the site of transcription. Mol. Cell 8, 33–43 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Naeger, L.K., Schoborg, R.V., Zhao, Q., Tullis, G.E. & Pintel, D.J. Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product. Genes Dev. 6, 1107–1119 (1992).

    Article  CAS  PubMed  Google Scholar 

  41. Lytle, J.R. & Steitz, J.A. Premature termination codons do not affect the rate of splicing of neighboring introns. RNA 10, 657–668 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dietz, H.C. & Kendzior, R.J., Jr. Maintenance of an open reading frame as an additional level of scrutiny during splice site selection. Nat. Genet. 8, 183–188 (1994).

    Article  CAS  PubMed  Google Scholar 

  43. Li, B. et al. Stop codons affect 5′ splice site selection by surveillance of splicing. Proc. Natl. Acad. Sci. USA 99, 5277–5282 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang, J., Chang, Y.F., Hamilton, J.I. & Wilkinson, M.F. Nonsense-associated altered splicing: a frame-dependent response distinct from nonsense-mediated decay. Mol. Cell 10, 951–957 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Wang, J., Hamilton, J.I., Carter, M.S., Li, S. & Wilkinson, M.F. Alternatively spliced TCR mRNA induced by disruption of reading frame. Science 297, 108–110 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Gersappe, A. & Pintel, D.J. A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner. Mol. Cell. Biol. 19, 1640–1650 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang, J. & Wilkinson, M.F. Site-directed mutagenesis of large (13-kb) plasmids in a single-PCR procedure. Biotechniques 29, 976–978 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Wang, J., Gudikote, J.P., Olivas, O.R. & Wilkinson, M.F. Boundary-independent polar nonsense-mediated decay. EMBO Rep. 3, 274–279 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Parks, D.R. & Herzenberg, L.A. Fluorescence-activated cell sorting: theory, experimental optimization, and applications in lymphoid cell biology. Methods Enzymol. 108, 197–241 (1984).

    Article  CAS  PubMed  Google Scholar 

  50. Chen, C.A. & Okayama, H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632–638 (1988).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Y.-J. Liu and Y.-H. Wang for technical advice on FACS analysis, A. Bhalla for technical assistance and G. Cote for useful comments. This study was supported by the US National Institutes of Health grant GM058595 and the Kleberg Fund for Innovative Research Program Allocation for IRG (M.D. Anderson Cancer Center).

Author information

Authors and Affiliations

  1. Department of Immunology, University of Texas M.D. Anderson Cancer Center, Houston, 77030, Texas, USA

    Jayanthi P Gudikote, J Saadi Imam, Ramon F Garcia & Miles F Wilkinson

Authors
  1. Jayanthi P Gudikote
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Saadi Imam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ramon F Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miles F Wilkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Miles F Wilkinson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Deletions in the VDJ exon do not affect NMD. (PDF 248 kb)

Supplementary Fig. 2

A mutation in the 3′ splice site of TCRβ IVS1 dramatically reduces the magnitude of downregulation in response to a PTC. (PDF 134 kb)

Supplementary Methods (PDF 50 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gudikote, J., Imam, J., Garcia, R. et al. RNA splicing promotes translation and RNA surveillance. Nat Struct Mol Biol 12, 801–809 (2005). https://doi.org/10.1038/nsmb980

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb980

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