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:

prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast

Nature Structural & Molecular Biology volume 14, pages 1077–1083 (2007)Cite this article

Abstract

Prp8 protein (Prp8p) is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 small nuclear ribonucleoproteins (snRNPs). Although it is ubiquitously expressed, mutations in the C terminus of human Prp8p cause the retina-specific disease retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks the mature U5 snRNP component Brr2p and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes that is disrupted by mutations that cause human RP.

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: Functional analysis of a nuclear localization signal in Prp8p.
Figure 2: More Prp8p–Aar2p–U5 snRNP complex forms with ΔNLS-Prp8p.
Figure 3: Analysis of Brr2p interactions.
Figure 4: Effects of prp8-rp mutations.
Figure 5: Cartoon showing the proposed pathway for U5 snRNP biogenesis in budding yeast.

Similar content being viewed by others

References

  1. Will, C.L. & Lührmann, R. Spliceosomal UsnRNP biogenesis, structure and function. Curr. Opin. Cell Biol. 13, 290–301 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Jurica, M.S. & Moore, M.J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. McKie, A.B. et al. Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13). Hum. Mol. Genet. 10, 1555–1562 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Vithana, E.N. et al. A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11). Mol. Cell 8, 375–381 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Chakarova, C.F. et al. Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa. Hum. Mol. Genet. 11, 87–92 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Keen, T.J. et al. Mutations in a protein target of the Pim-1 kinase associated with the RP9 form of autosomal dominant retinitis pigmentosa. Eur. J. Hum. Genet. 10, 245–249 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Maita, H. et al. PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor. Exp. Cell Res. 300, 283–296 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Kiss, T. Biogenesis of small nuclear RNPs. J. Cell Sci. 117, 5949–5951 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Bertrand, E. & Bordonne, R. Assembly and traffic of small nuclear RNPs. Prog. Mol. Subcell. Biol. 35, 79–97 (2004).

    Article  PubMed  Google Scholar 

  10. Yong, J., Wan, L. & Dreyfuss, G. Why do cells need an assembly machine for RNA-protein complexes? Trends Cell Biol. 14, 226–232 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Huber, J. et al. Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure. EMBO J. 17, 4114–4126 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jantsch, M.F. & Gall, J.G. Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte. J. Cell Biol. 119, 1037–1046 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Romac, J.M.J., Graff, D.H. & Keene, J.D. The U1 small nuclear ribonucleoprotein (snRNP) 70k protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain. Mol. Cell. Biol. 14, 4662–4670 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hetzer, M. & Mattaj, I.W. An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B′′ spliceosome proteins. J. Cell Biol. 148, 293–304 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nesic, D., Tanackovic, G. & Kramer, A. A role for Cajal bodies in the final steps of U2 snRNP biogenesis. J. Cell Sci. 117, 4423–4433 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Ohno, M., Segref, A., Bachi, A., Wilm, M. & Mattaj, I.W. PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell 101, 187–198 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Grainger, R.J. & Beggs, J.D. Prp8 protein: at the heart of the spliceosome. RNA 11, 533–557 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jackson, S.P., Lossky, M. & Beggs, J.D. Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing. Mol. Cell. Biol. 8, 1067–1075 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Luo, H.R., Moreau, G.A., Levin, N. & Moore, M.J. The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes. RNA 5, 893–908 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Martinez-Gimeno, M. et al. Mutations in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in Spanish families with autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 44, 2171–2177 (2003).

    Article  PubMed  Google Scholar 

  21. Kondo, H. et al. Diagnosis of autosomal dominant retinitis pigmentosa by linkage-based exclusion screening with multiple locus-specific microsatellite markers. Invest. Ophthalmol. Vis. Sci. 44, 1275–1281 (2003).

    Article  PubMed  Google Scholar 

  22. Ziviello, C. et al. Molecular genetics of autosomal dominant retinitis pigmentosa (ADRP): a comprehensive study of 43 Italian families. J. Med. Genet. 42, e47 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Testa, F. et al. Clinical phenotype of an Italian family with a new mutation in the PRPF8 gene. Eur. J. Ophthalmol. 16, 779–781 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. De Erkenez, A.C., Berson, E.L. & Dryja, T.P. Novel mutations in the PRPC8 gene, encoding a pre-mRNA splicing factor in patients with autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 43 e-abstract 791 (2002).

  25. Gottschalk, A., Kastner, B., Lührmann, R. & Fabrizio, P. The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p. RNA 7, 1554–1565 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Stevens, S.W. et al. Biochemical and genetic analysis of the U5, U6, and U4/U6.U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae. RNA 7, 1543–1553 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Umen, J.G. & Guthrie, C. A novel role for a U5 snRNP protein in 3′ splice site selection. Genes Dev. 9, 855–868 (1995).

    Article  CAS  PubMed  Google Scholar 

  28. Bordonne, R. Functional characterization of nuclear localization signals in yeast Sm proteins. Mol. Cell. Biol. 20, 7943–7954 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Achsel, T., Ahrens, K., Brahms, H., Teigelkamp, S. & Lührmann, R. The human U5–220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Mol. Cell. Biol. 18, 6756–6766 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. van Nues, R.W. & Beggs, J.D. Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes. Genetics 157, 1451–1467 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Pena, V., Liu, S., Bujnicki, J.M., Lührmann, R. & Wahl, M.C. Structure of a multipartite protein-protein interaction domain in splicing factor prp8 and its link to retinitis pigmentosa. Mol. Cell 25, 615–624 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Fischer, U. et al. Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport. J. Cell Biol. 113, 705–714 (1991).

    Article  CAS  PubMed  Google Scholar 

  33. Boon, K.-L., Norman, C.M., Grainger, R.J., Newman, A.J. & Beggs, J.D. Prp8p dissection reveals domain structure and protein interaction sites. RNA 12, 198–205 (2005).

    Article  PubMed  Google Scholar 

  34. Steinberg, R.H. Monitoring communications between photoreceptors and pigment epithelial cells: effects of “mild” systemic hypoxia. Friedenwald lecture. Invest. Ophthalmol. Vis. Sci. 28, 1888–1904 (1987).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  36. Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Puig, O., Gottschalk, A., Fabrizio, P. & Seraphin, B. Interaction of the U1 snRNP with nonconserved intronic sequences affects 5′ splice site selection. Genes Dev. 13, 569–580 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lesser, C.F. & Guthrie, C. Mutational analysis of pre-messenger RNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics 133, 851–863 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Niedenthal, R.K., Riles, L., Johnston, M. & Hegemann, J.H. Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast 12, 773–786 (1996).

    Article  CAS  PubMed  Google Scholar 

  40. Carter, K.C. et al. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science 259, 1330–1335 (1993).

    Article  CAS  PubMed  Google Scholar 

  41. Samarsky, D.A., Fournier, M.J., Singer, R.H. & Bertrand, E. The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization. EMBO J. 17, 3747–3757 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Olson, B.L. & Siliciano, P.G. A diverse set of nuclear RNAs transfer between nuclei of yeast heterokaryons. Yeast 20, 893–903 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Lin, R.-J., Newman, A.J., Cheng, S.-C. & Abelson, J. Yeast mRNA splicing in vitro. J. Biol. Chem. 260, 14780–14792 (1985).

    CAS  PubMed  Google Scholar 

  44. Boon, K.-L. et al. Yeast Ntr1/Spp382 mediates Prp43 function in postspliceosomes. Mol. Cell. Biol. 26, 6016–6023 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bartels, C., Urlaub, H., Lührmann, R. & Fabrizio, P. Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing. J. Biol. Chem. 278, 28324–28334 (2003).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P. Fabrizio (Max Planck Institute, Göttingen) for the very generous gift of anti-Aar2p and anti-Snu114p antibodies, O. Cordin (University of Edinburgh) for anti-Brr2p anti-peptide antibodies, M. Spiller for assistance with immunofluorescence staining and microscopy, A. Kutach and C. Guthrie for sharing information before publication, and I. Davis, M. Reijns and D. Tollervey for critical comments. This work was funded by studentships from The Darwin Trust of Edinburgh to K.-L.B. and T.A. and by Wellcome Trust Grants 067311 (J.D. Beggs) and 073988 (C.F.I.) and European Commission grant LSH-2004-518238 (EURASNET Network of Excellence). J.D. Beggs is the Royal Society Darwin Trust Professor.

Author information

Author notes

  1. Kum-Loong Boon

    Present address: Present address: Center for Molecular Neurobiology, The Ohio State University, 132 Rightmire Hall, 1060 Carmack Road, Columbus, Ohio 43210, USA.,

Authors and Affiliations

  1. Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK

    Kum-Loong Boon, Richard J Grainger, Parastoo Ehsani, J David Barrass, Tatsiana Auchynnikava & Jean D Beggs

  2. Section of Ophthalmology and Neuroscience, Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Beckett Street, Leeds, LS9 7TF, UK

    Chris F Inglehearn

Authors
  1. Kum-Loong Boon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard J Grainger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Parastoo Ehsani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J David Barrass
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tatsiana Auchynnikava
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chris F Inglehearn
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jean D Beggs
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.-L.B. contributed Figures 1, 2, 4c, 4e–h and 5 and Supplementary Figures 1, 3 and 6; R.J.G. contributed Figures 3b, 3c and 4b and Supplementary Figure 2; P.E. contributed Figure 4d and investigated the effects of HA-tagging Prp8p with and without the rp mutations (data not shown); J.D. Barrass performed the RT-PCR and microarray analyses (Supplementary Figs. 4 and 5); T.A. contributed Figure 3a; C.F.I. provided expertise on RP; J.D. Beggs supervised all the work and wrote most of the paper.

Corresponding author

Correspondence to Jean D Beggs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 681 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boon, KL., Grainger, R., Ehsani, P. et al. prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nat Struct Mol Biol 14, 1077–1083 (2007). https://doi.org/10.1038/nsmb1303

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1303

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