Ian C Eperon

Personal Details

Ian C EperonChair in Biochemistry
Ian Eperon Headshot 2.1

DepartmentMolecular and Cell Biology
Telephone: +44 (0)116 229 7012
Emaileci@le.ac.uk
Address: Lancaster Rd, LE1 7HB
Web Links:

Biography

I studied Biochemistry at the University of Bristol, from 1974 to 1977. The department had considerable strengths in enzyme kinetics, crystallography, nucleic acids and bioenergetics, all of which shaped my view of biochemistry as an essentially molecular and quantitative science. My PhD research was done with Fred Sanger at the MRC LMB in Cambridge, starting just as he was publishing the dideoxy sequencing method for DNA that would revolutionize biological science. Most modern sequencing methods still rely on the principles of using extension by DNA polymerase from a primer with a defined 5’ terminus, established in Sanger's papers in 1975 and 1977. I worked on cloning and sequencing human, bovine and trypanosome mitochondrial genomes. It was slow work; nowadays the sequences of these genomes are determined routinely for deducing ancestry and relationships and even for the definitive identification of the source of processed meat.

I obtained a fellowship in 1981 to work with Joan Steitz at Yale University on the newly-discovered process known as RNA splicing. In 1984, I was appointed as a lecturer in Leicester. This was followed later by appointments as a Reader and then Professor of Biochemistry. I have worked on many aspects of pre-mRNA splicing, but the main focus has always been the mechanisms by which splice sites are selected in mammals. In 2004, I collaborated with Clive Bagshaw to start single molecule studies of RNA splicing, since it had long been apparent to me that conventional methods could not adequately address mechanistic questions in nuclear extracts, which are the only functional in vitro system. Dmitry Cherny, in our group, was the first to apply single molecule multicolour colocalization to analyse protein binding in nuclear extracts, and the new insights this enables when coupled with innovative chemical approaches are my main current research interest. This programme of research is interdisciplinary and is a collaboration with Andrew Hudson (Chemistry) and Glenn Burley (Chemistry, University of Strathclyde).

We applied our nascent understanding of splicing in early experiments to shift splicing for therapeutic purposes in muscular dystrophy and then spinal muscular atrophy. This led to an interest in the formation and roles of RNA quadruplexes, and to a collaboration with Cyril Dominguez (in this department) to investigate ways of exploiting quadruplexes to shift the splicing of some genes such that, instead of producing anti-apoptotic proteins, they produce pro-apoptotic proteins. The intention is to develop these as potential adjunct therapies in cancer.

Qualifications

  • BSc with First Class Honours, and Faculty of Science scholarship, University of Bristol, 1977
  • PhD, University of Cambridge, 1981

Publications

  1. Eperon, I.C., Anderson, S. and Nierlich, D.P. (1980). Distinctive sequence of human mitochondrial ribosomal RNA genes. Nature 286, 460-467.
  2. Barrell, B. G., Anderson, S., Bankier, A. T., de Bruijn, M. H., Chen, E., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J., Staden, R., and Young, I. G. (1980). Different pattern of codon recognition by mammalian mitochondrial tRNAs. Proc. Nat. Acad. Sci. USA 77, 3164-3166. (authors in alphabetical order after first)
  3. de Bruijn, M. H., Schreier, P. H., Eperon, I. C., Barrell, B. G., Chen, E. Y., Armstrong, P. W., Wong, J. F., and Roe, B. A.(1980). A mammalian mitochondrial serine transfer RNA lacking "dihydrouridine" loop and stem. Nucl. Acids Res. 8, 5213-5222
  4. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J., Staden, R., and Young, I. G.(1981). The sequence and organisation of the human mitochondrial genome. Nature 290, 457-467. (authors in alphabetical order)
  5. Anderson, S., de Bruijn, M. H., Coulson, A. R., Eperon, I. C., Sanger, F., and Young, I. G. (1982). The complete sequence of bovine mitochondrial DNA: Conserved features of the mammalian mitochondrial genome. J. Mol. Biol. 156, 683-717. (authors in alphabetical order)
  6. Eperon, I.C., Janssen, J.W.G., Hoeijmakers, J.H.J. and Borst, P. (1983). The major transcripts of the kinetoplast DNA of Trypanosoma brucei are very small ribosomal RNAs. Nucl. Acids Res. 11, 105-125.

Publications from my Laboratory in Leicester

  1. Eperon, L.P., Estibeiro, J.P. and Eperon, I.C. (1986). The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA. Nature 324, 280-282.
  2. Eperon, I.C. (1986) Rapid preparation of bacteriophage DNA for sequence analysis in sets of 96 clones, using filtration. Analyt. Biochem. 156, 406-412.
  3. Skinner, J.A. and Eperon, I.C. (1986). Misincorporation by AMV reverse transcriptase shows strong dependence on the combination of template and substrate nucleotides. Nucl. Acids Res. 14, 6945-6964.
  4. Eperon, I.C. (1986). M13 vectors with T7 polymerase promoters : transcription limited by oligonucleotides. Nucl. Acids Res. 14, 2830.
  5. Price, G.J., Jones, P., Davison, M.D., Patel, B., Eperon, I.C. and Critchley, D.R. (1987). Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts. Biochem. J. 245, 595-603.
  6. Griffiths, A.D., Potter, B.V.L. and Eperon, I.C. (1987). Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucl. Acids Res. 15, 4145-4162.
  7. Deeney, C.M.M., Eperon, I.C. and Potter, B.V.L. (1987). Self-splicing of tetrahymena rRNA can proceed with phosphorothioate substitution at the splice sites. Nucl. Acids Res. Symposium Series No. 18, 277-280.
  8. Turnbull-Ross, A.D., Else, A.J. and Eperon, I.C. (1988). The dependence of splicing efficiency on the length of 3' exon. Nucl. Acids Res. 16, 395-411.
  9. Eperon, I.C. (1988). M13 cloning and dideoxy sequencing. In Gene Cloning and Analysis: a Laboratory Guide (ed. G.J. Boulnois), pp. 107-122 Blackwell Scientific. Eperon, I.C. (1988) Oligonucleotide Mutagenesis. Ibid., pp. 123-128. Eperon, I.C. (1988) Mapping RNA. Ibid., pp. 129-136.
  10. Griffiths, A.D., Potter, B.V.L. and Eperon, I.C. (1988). Substitution of pre-mRNA with phosphorothioate linkages reveals a new splicing-related reaction. J. Biol. Chem. 263, 12295-12304.
  11. Eperon, L.P., Graham, I.R., Griffiths, A.D. and Eperon, I.C. (1988). The effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell 54, 393-401.
  12. Lear, A.L., Eperon, L.P., Wheatley, I.M. and Eperon, I.C. (1990). A hierarchy for 5' splice site preference determined in vivo. J. Mol. Biol. 211, 103-115.
  13. Cunningham, S.A., Else, A.J., Potter, B.V.L., and Eperon, I.C. (1991). The influences of separation and adjacent sequences on the use of alternative 5' splice sites. J. Mol. Biol. 217, 265-281.
  14. Skilton, H., Eperon, I.C., and Rivett, J.A. (1991). Co-purification of a small RNA species with multicatalytic proteinase (proteasome) from rat liver. FEBS Lett. 279, 351-355.
  15. Garde, J., Bell, S.C., and Eperon, I.C. (1991). Multiple forms of mRNA encoding human pregnancy-associated endometrial alpha 2-globulin, a beta-lactoglobulin homologue. Proc. Natl. Acad. Sci. USA 88, 2456-2460.
  16. Hamshere, M., Dickson, G., and Eperon, I. (1991). The muscle-specific domain of mouse N-CAM: structure and alternative splicing patterns. Nuc. Acids Res. 19, 4709-4716.
  17. Waites, G.T., Graham, I.R., Jackson, P., Millake, D.B., Patel, B., Blanchard, A.D., Weller, P., Eperon, I.C., and Critchley, D.R. (1991). Mutually-exclusive splicing of calcium-binding domain exons in chick alpha-actinin. J. Biol. Chem. 267, 6263-6271.
  18. Graham, I.R., Hamshere, M., and Eperon, I.C. (1992). Alternative splicing of a human alpha-tropomyosin muscle-specific exon: Identification of determining sequences. Mol. Cell. Biol. 12, 3872-3882.
  19. Hamshere, M., and Eperon, I.C. (1993) Applications of gene transfer for analysis of pre-mRNA splicing. Journal of Tissue Culture Methods 15, 99-107.
  20. Eperon, I.C., Ireland, D.C., Smith, R.A., Mayeda, A., and Krainer, A.R. (1993). Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 12, 3607-3617.
  21. Eperon, I.C. and Krainer, A.R. (1994). Splicing of Messenger RNA Precursors. In RNA Processing: a Practical Approach, eds Hames, D., and Higgins, S. Oxford University Press, pp. 57-101.
  22. Willmott, C.J.R., Critchlow, S.E., Eperon, I.C., and Maxwell, A. (1994). The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase. J. Mol. Biol. 242, 351-363.
  23. Vekony, M.A., Holder, J.E., Lee, A.I., Horrocks, C., Eperon, I.C., & Camp, R.D.R. (1997). Selective simplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping. J. Invest. Dermatol. 109, 5-13.
  24. Dunckley, M., Eperon, I.C., & Dickson, G. (1997). Modulation of splicing in the DMD gene by antisense oligonucleotides. Nucleosides and Nucleotides 16, 1665-1668.
  25. Elliott, D.J., Oghene, K., Makarov, G., Makarova, O., Hargreave, T.B., Chandley, A.C., Eperon, I.C., & Cooke, H.J. (1998). Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development. J. Cell Sci. 111, 1255-1265.
  26. Dunckley, M.G., Manoharan, M., Villiet, P., Eperon, I.C., and Dickson, G. (1998). Modification of splicing in the dystrophin gene in cultured mdx muscle cells by antisense oligoribonucleotides. Hum. Mol. Gen. 7, 1083-1090.
  27. O’Mullane, L., & Eperon, I.C. (1998). The pre-mRNA 5’ cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5’ splice sites. Mol. Cell. Biol. 18, 7510-7520.
  28. Venables, J.P., & Eperon, I.C. (1999). The roles of RNA-binding proteins in spermatogenesis and male infertility. Curr. Op. Gen. Dev. 9, 346-354.
  29. Frantz, S.A., Thiara, A.S., Lodwick, D., Eperon, I.C., & Samani, N.J. (1999). Exon repetition in mRNA. Proc. Natl Acad. Sci. USA 96, 5400-5405.
  30. Venables, J.P., Vernet, C., Chew, S.L., Elliott, D.J., Cowmeadow, R.B., Wu, J., Cooke, H.J., Artzt, K., and Eperon, I.C. (1999). T-STAR/ÉTOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. Hum. Mol. Gen. 8, 959-969.
  31. Chew, S.L., Baginsky, L., and Eperon, I.C. (2000). An exonic splicing silencer in the testis-specific DNA ligase III beta exon. Nucleic Acids Res. 27, 402-410.
  32. Venables, J.P., Elliott, D.J., Makarova, O.V., Makarov, E.M., Cooke, H.J,.& Eperon, I.C. (2000). RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2b and affect splicing. Hum. Mol. Gen. 9, 685-694.
  33. Elliott, D.J., Venables, J.P., Newton, C., Lawson, D., Boyle, S., Eperon, I.C., & Cooke, H.J. (2000). An evolutionarily conserved germ cell-specific hnRNP is encoded by a retrotransposed gene. Hum. Mol. Gen. 9, 2117-2124. (September 2000, front cover).
  34. Eperon, I.C., Makarova, O., Mayeda, A., Munroe, S.H., Cáceres, J., Hayward, D.G., & Krainer, A.R. (2000) Selection of alternative 5' splice sites: the role of U1 snRNP and models for the antagonistic affects of SF2/ASF and hnRNP A1. Mol. Cell. Biol. 20, 8303-8318.
  35. Nasim, M.T., Chowdhury, H.M., & Eperon, I.C. (2002). A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells. Nuc. Acids Res. 30, e109 (6 pages).
  36. Stover, C.M., Lynch, N.J., Dahl, M.R., Hanson, S., Takahashi, M., Frankenberger, M., Ziegler-Heitbrock, L., Eperon, I.C., Thiel, S., & Schwaeble, W.J. (2003). Murine serine proteases MASP-1 and MASP-3, components of the lectin pathway activation complex of complement, are encoded by a single structural gene. Genes and Immunity 4, 374-384.
  37. Skordis, L.A., Dunckley, M.G., Yue, B.-G., Eperon, I.C., and Muntoni, F. (2003). Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc. Natl Acad. Sci. USA 100, 4114-4119.
    Reviewed in: 
    http://www.nature.com/horizon/rna/background/splice.html
    http://www.nature.com/horizon/rna/highlights/s1_spec1.html
    http://www.biomedcentral.com/news/20030926/07
  38. Nasim, M.T., Chernova, T.K., Chowdhury, H.M., Yue, B.-G. and Eperon, I.C. (2003). HnRNP G and Tra2b: opposite effects on splicing matched by antagonism in RNA binding. Hum. Mol. Gen. 12, 1337-1348.
  39. Eperon, I.C., and Muntoni, F. (2003). Response to Buratti et al.: Can a 'patch' in a skipped exon make the pre-mRNA splicing machine run better? Trends Mol. Med. 9, 233-234.
  40. Rigatti, R., Jia, J.-H., Samani, N.J., and Eperon, I.C. (2004). Exon repetition: a major pathway for processing mRNA of some genes is allele-specific. Nuc. Acids Res. 32, 441-446.
  41. Nasim, M.T., Eperon, I.C., Wilkins, B.M., & Brammar, W.J. (2004). The activity of a single-stranded promoter of plasmid ColIb-P9 depends on its secondary structure. Molecular Microbiology 53, 405-417.
  42. Dixon, R.J., Eperon, I.C., Hall, L., & Samani, N.J. (2005). A genome-wide survey demonstrates widespread non-linear mRNA in expressed sequences from multiple species. Nuc. Acids Res. 33, 5904-5913.
  43. Nasim, M.T., and Eperon, I.C. (2006). A double-reporter splicing assay for determining splicing efficiency in mammalian cells. Nature Protocols 1, 1022-1028.
  44. Dixon, R.J., Eperon, I.C., & Samani, N.J. (2007). Complementary intron sequence motifs associated with human exon repetition: a role for intragenic, inter-transcript interactions in gene expression. Bioinformatics 23, 150-155.
  45. Malygin, A., Parakhnevitch, N.M., Ivanov, A.V., Eperon, I.C., & Karpova, G.G. (2007). Human ribosomal protein S13 regulates expression of its own gene at the splicing step by a feedback mechanism. Nuc. Acids Res. 35, 6414-6423.
  46. Cherny, D.I., Eperon, I.C., & Bagshaw, C.R. (2009). Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes. Eur. Biophys. J. 38, 395-405.
  47. Cherny, D., Gooding, C., Eperon, G.E., Coelho, M.B., Bagshaw, C.R., Smith, C.W.J., & Eperon, I.C. (2010). Stoichiometry of a regulatory splicing complex revealed by single molecule analyses. EMBO J. 29, 2161-2172.
  48. Ghigna, C., De Toledo, M., Valacca, C., Bonomi, S., Gallo, G., Apicella, M., Eperon, I., Jamal Tazi, J., and Biamonti, G. (2010). Pro-metastatic splicing of Ron proto-oncogene mRNA can be reversed: therapeutic potential of bifunctional oligonucleotides and indoles. RNA Biology 7, 4, 1-9.
  49. Owen, N., Zhou, H., Malygin, A., Sangha, J., Smith, L.D., Muntoni, F., & Eperon, I.C. (2011). Design principles for bifunctional targeted oligonucleotide enhancers of splicing. Nucleic Acids Res. 39, 7194-7208.
  50. Fedorova, O.A., Moiseeva, T.N., Nikiforov, A.A., Tsimokha, A.S., Livinskaya, V.A., Hodson, M., Bottrill, A., Evteeva, I.N., Ermolayeva, J.B., Kuznetzova, I.M., Turoverov, K.K., Eperon, I., and Barlev, N.A. (2011). Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism. Biochem. Biophys. Res. Comm. 416, 258-265.
  51. Hodson, M.J., Hudson, A.J., Cherny, D., & Eperon, I.C. (2012). The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites. Nucleic Acids Res. 40, 6850-6862. Featured article (top 5 %)
  52. Kafasla, P., Mickleburgh, I., Llorian, M., Coelho, M., Gooding, C., Cherny, D., Joshi, A., Kotik-Kogan, O., Curry, S., Eperon, I., Jackson, R., & Smith, C.W.J. (2012). Defining the roles and interactions of PTB. Biochem. Soc. Trans. 40, 815-820.
  53. Eperon, I. (2012). New ways to nudge splicing. Nature Chemical Biology 8, 507-508.
  54. Lewis, H., Perrett, A.J., Burley, G.A., & Eperon, I.C. (2012). An RNA splicing enhancer that does not act by looping. Angewandte Chem. Int. Ed. 51, 9800-9803
  55. Perrett, A.J., Dickinson, R.L., Krpetić, Z., Brust, M., Lewis, H., Eperon, I.C.* & Burley, G.A.* (2012). Conjugation of PEG and gold nanoparticles to increase the accessibility and valency of tethered RNA splicing enhancers. Chemical Science 4, 257-265
  56. Roca, X., Krainer, A.R., & Eperon, I.C. (2013). Pick one, but be quick: 5’ splice sites and the problems of too many choices. Genes & Development 27, 129-144
  57. Zhou, H., Janghra, N., Mitrpant, C., Dickinson, R., Anthony, K., Price, L., Eperon, I., Wilton, S., Morgan, J., & Muntoni, F. (2013). A novel morpholino oligomer targeting ISS-N1 improves rescue of severe SMA transgenic mice. Human Gene Therapy 24, 1-12
  58. Gooding, C., Edge, C., Lorenz, M., Coelho, M., Winters, W., Kaminski, C., Cherny, D., Eperon, I.C., & Smith, C.W.J. (2013). MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3. Nucleic Acids Res. 41, 4765-4782
  59. Schmid, R., Grellscheid, S.N., Ehrmann, I., Dalgleish, C., Danilenko, M., Paronetto, M.P., Pedrotti, S., Grellscheid, D., Dixon, R.J., Sette, C., Eperon, I.C., & Elliott, D.J. (2013). The splicing landscape is globally reprogrammed during male meiosis. Nucleic Acids Res. 41, 10170-10184
  60. Smith, L.D., Lucas, C.M., and Eperon, I.C. (2013). Intron retention in the alternatively spliced region of RON results from weak 3’ splice site recognition. PLoS ONE 8(10): e77208. .
  61. Smith, L.D., Dickinson, R.L., Lucas, C.M., Cousins, A., Malygin, A.A., Weldon, C., Perrett, A.J., Bottrill, A.R., Searle, M.S., Burley, G.A., & Eperon, I.C. (2014). A targeted oligonucleotide enhancer of SMN2 exon 7 splicing forms competing quadruplex and protein complexes in functional conditions. Cell Reports 9, 193-205.
  62. Weinmeister, R., Freeman, E., Eperon, I.C., Stuart, A.M., & Hudson, A.J. (2015). Single-fluorophore detection in femtoliter droplets generated by flow focusing. ACS Nano 9, 9718-9730
  63. Feracci, M., Foot, J.N., Grellscheid, S.N., Danilenko, M., Stehle, R., Kang, H.S., Dalgleish, C., Meyer, N.H., Liu, Y., Sattler, M., Eperon, I.C., Elliott, D.J., & Dominguez, C. (2016). Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68. Nat. Commun. 7, 10355 .
  64. Weldon, C., Eperon, I.C., and Dominguez, C. (2016). Do we know whether potential G-quadruplexes actually form in long functional RNA molecules? Biochemical Society Transactions 15, 1761-1768.
  65. Weldon, C., Behm-Ansmant, I., Hurley, L.H., Burley, G.A., Branlant, C., Eperon, I.C.,* and Dominguez, C.* (2017). Identification of G-quadruplexes in functional RNAs using 7-deaza-guanine. Nature Chemical Biology 13, 18-20 (* Joint senior authors).
  66. Reichenbach, L.F., Agnew, C., Ahmad Sobri, A., Burton, N., Zaccai, N.R., Eperon, L.P., de Ornellas, S., Eperon, I.C., Brady, R.L., and Burley, G.A. (2016). Structural Basis for mis-pairing of an artificially expanded genetic information system. Chem (Cell Press) 1, 946-958.
  67. Chen, L., Weinmeister, R., Kralovicova, J., Eperon, L.P., Vorechovsky, I., Hudson, A.J., and Eperon, I.C. (2017). Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways. Nucleic Acids Research 45, 2051-2067
  68. Weldon, C., Dacanay, J.G., Gokhale, V., Behm-Ansmant, I., Burley, G.A., Branlant, C., Hurley, L.H., Dominguez, C., & Eperon, I.C. (2017). Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X. Nucleic Acids Research 46, 886-896.
  69. Jobbins, A.M., Reichenbach, L.F., Lucas, C.M., Hudson, A.J., Burley, G.A., & Eperon, I.C. (2018). The mechanisms of a mammalian splicing enhancer. Nucleic Acids Research 46, 2145-2158. Designated as an NAR Breakthrough article.
  70. Jobbins, A.M., Weinmeister, R., Lucas, C.M., Chen, L., Cléry, A., Eperon, L.P., Hodson, M.J., Alfi, A., Hudson, A.J., and Eperon, I.C. (2018). U1 snRNPs mediate exon-independent recruitment of single molecules of SRSF1. In revision.
  71. Reichenbach, L.F., Jobbins, A.M., Eperon, I.C., & Burley, G.A. (2018). Synthesis of RNA splicing constructs containing a sequence of non-RNA building blocks using splint ligation. In preparation

Research

  • Mechanisms of selection of splice sites in pre-mRNA (see Biography and publications)
  • Understanding and exploiting the actions of RNA G-quadruplexes in pre-mRNA splicing (with Dr C. Dominguez).
  • Use of single molecule multicolour colocalization fluorescence microscopy to study reactions and development of new single molecule methods (with Dr A.J. Hudson); use of novel chemical biology tools to study RNA-protein dynamics and interactions (with Dr G.A. Burley, Strathclyde).

RNA Splicing

Single molecules to therapy

Almost all mammalian genes produce multiple isoforms of mRNA and protein by alternative splicing; this, not the number of genes, is the key to the evolution of complex eukaryotes. We are interested in the molecular mechanisms by which sites are selected and have made a number of contributions

The very existence of extensive alternative splicing in mammals tells us something important: the splice site sequences are not very information-rich, and there are lots of candidate sites in the pre-mRNA. Some are used constitutively, some in specific circumstances, some only if the normal site is mutated, and others never. How is the RECOGNITION by specific factors turned into SELECTION of a site? Most nucleotides in or near some exons contribute to the use of an exon, presumably because they are bound by one or more of the many RNA-binding proteins that appear to compete for binding, with low and overlapping specificities. To understand recognition and selection, we need to understand what the pre-mRNA looks like: which proteins are bound, where, and in what numbers, and what effects they have on the behaviour of the RNA.

It became clear to me some years ago that splicing was too complicated for conventional molecular approaches to be very informative, and, in collaboration with Clive Bagshaw, we began a programme to develop single molecule methods for analysing splicing in nuclear extracts. Dmitry Cherny in this lab did the first single molecule experiments on mammalian splicing and showed that we could determine the numbers of regulatory proteins associated with each molecule of RNA in functional conditions (nuclear extracts). We are now addressing some of the most intractable problems regarding splice site selection and the mechanisms of action of proteins that regulate this process, in collaboration with Andrew Hudson (Leicester), Cyril Dominguez (Leicester) and Glenn Burley (Strathclyde).

Recent Highlights

SRSF1 (Jobbins et al., 2018), in collaboration with Glenn Burley (Strathclyde) and Andrew Hudson (Leicester)

We have recently published an analysis of the way in which the splicing factor SRSF1 engages with enhancer sequences when it stimulates splicing. This was awarded 'Breakthrough' status by Nucleic Acids Research, and the text of the notice is reproduced here.

A collaborative research team led by Drs. Glenn Burley, Andrew Hudson and Ian Eperon at the University of Strathclyde and University of Leicester (UK) has described studies that provide important new insight into the mechanisms that regulate pre-mRNA splicing in mammalian cells.

Exonic splicing enhancers (ESEs) are sequences that play key roles in determining whether the exons in which they reside are incorporated into the mature mRNA.  They were discovered and characterized in the mid-1990's, when it was demonstrated that canonical ESEs are bound by SR proteins (the prototype of which is SRSF1) to activate particular splicing patterns. Based largely on elegant experiments with Drosophila enhancers performed 20 years ago by the Maniatis lab, this process is generally thought to involve stable binding of the SR protein to an ESE, followed by rate-limiting interactions with factors bound at nearby splice sites.  However, it is difficult to reconcile this model with the recently discovered density of regulatory sequences in mammalian exons, and there have been no molecular tests that might resolve this discrepancy.

In this study, the investigators employ single molecule multi-colour colocalization to quantify stable SRSF1 binding in a nuclear extract that supports splicing, and chemical biology to dissect the subsequent activation process.  The results demonstrate that SRSF1-dependent mammalian enhancers do not follow the Drosophila paradigm, thereby breaking with traditional views of ESE function. Specifically, the authors conclude that the initial binding of SRSF1 is weak, and that interactions involving 3D diffusion incorporate a single molecule of SRSF1.  The picture that emerges is one in which exons do not form stable complexes with SR proteins that inevitably lead to splice site usage.  Instead, there appears to be a dynamic interplay among regulatory proteins that bind only transiently to the RNA, and the outcome depends on the chance that an SR protein is occupying an ESE at a particular moment, such as when the ESE is close to the 3’ splice site.

These findings require a profound change in the way in which the mechanisms of alternative splicing are studied.  Hitherto, both in vitro and in vivo, the emphasis has been on ‘what’ and ‘where’:  which regulatory proteins bind where on the pre-mRNA. Now, however, ‘when’ will have to be added:  it will be important to find the probability of binding, the lifetime of the occupied state, the numbers and combinations of proteins bound concurrently and the probabilities of interactions with target splice sites.  Moreover, in vivo, this dynamic flux is superimposed on the ways in which transcription itself affects the times of binding of regulatory factors, as first proposed by Eperon thirty years ago.  Addressing these challenges is a formidable task for the years to come.

Reviewers and editors familiar with the work have stated that the study "re-addresses long-standing open questions about ESE action using approaches that would have been impossible twenty years ago ", that the results are "unexpected and contrast with the interpretation of (historical) data" and that the work "provides interesting new insights that could not have been obtained by conventional ensemble methods and that will be of wide interest in the field of splicing regulation and its mechanisms."

The University of Strathclyde is the second oldest university in Glasgow, Scotland and one of the UK's first technological universities by Royal Charter.  The University of Leicester is a public university founded in the early 20th century that has recently created the Leicester Institute for Structural and Chemical Biology, to which Hudson and Eperon belong.  The Burley, Hudson and Eperon laboratories study the chemistry, biology and mechanism of regulated RNA splicing, among other topics.  The research project was supported by the Leverhulme Trust.

Nucleic Acids Research is a publication of Oxford University Press and is fully open access.  Its mission is to provide outstanding, scientist-led evaluation and dissemination of the highest caliber research across a wide range of disciplines focused on the role of nucleic acids and nucleic acid interacting molecules in cellular and molecular biology.  Breakthrough articles at NAR describe studies that solve a long-standing problem in their field, or provide exceptional new insight and understanding into an area of research that will clearly motivate and guide new research opportunities and directions.  They represent the top papers that NAR receives for publication, and are selected by the Editors based on nominations by authors and/or reviewers, and on the subsequent recommendation of the reviewers and editorial board members.

"The mechanisms of a mammalian splicing enhancer"

(Andrew M. Jobbins, Linus Reichenbach, Christian M. Lucas, Andrew J. Hudson, Glenn Burley and Ian C. Eperon; University of Strathclyde and University of Leicester)

ABSTRACT:

"Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors.  It was inferred from measurements of splicing around twenty years ago that Drosophiladoublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets.  However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise.  Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE.  We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3’ splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3’ splice site.  These insertions did not block activation, and we conclude that the activation step involves direct interactions.  These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site."

PTB (Cherny et al., 2010). In collaboration with Chris Smith and colleagues, Cambridge.

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of α-tropomyosin via distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts.

In collaboration with Chris Smith and colleagues at Cambridge, we tested these models using single molecule approaches. The number of bound PTB molecules was determined both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analyzing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multi-step process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity. See Cherny et al. (2010), EMBO J. 29, 2161-2172.

Cherny et al Fig 2.jpg

This is a representative image showing single molecules. The tropomyosin RNA was labelled with the fluorophore Cy5 by hybridising it to an oligonucleotide that contained both Cy5 and a biotin group for attachment to the streptavidin-derivatized surface of the silica slide. The RNA was incubated in nuclear extract made from cells expressing mEGFP-PTB, and then the reaction was diluted and injected onto the slide. The false colour image shows single molecules of RNA (Cy5, red) and mEGFP-PTB (green). Circles show co-localization. About half the PTB in the extract was labelled; the rest is endogenous. To work out how many molecules of GFP and hence of PTB were present on the RNA, Dmitry Cherny counted the number of steps in which the signal bleached and also measured the total emissions. From these results he calculated that the wild-type RNA was bound by 5-6 molecules of protein. Mutants RNA sequences bound fewer.

Next, we attempted to use this information to model the binding sites of the proteins on the RNA. Using structural information about the four RNA-binding domains of PTB (Oberstrass et al. (2005), Science 309, 2054-2057), three of which bind with some specificity, Giles Eperon identified possible sites for individual proteins, and then used gel shift data from Clare Gooding, Cambridge, to search for combinations permitting three proteins to bind upstream of the exon.

Cherny et al Fig 6 A_B.jpg

Each coloured line represents the region bound by a single protein molecule. We inferred that there were many ways in which a single protein with four RNA-binding domains could bind, but that it was possible that three proteins could be accommodated, again in a number of possible arrangements. Since each of the three domains of PTB that we took not account binds only two or three nucleotides with any specificity, it seems probable that the apparently very high affinity of PTB for the tropomyosin sequences is the result of the very large number of possible arrangements. If one protein binds, then interference with the binding of a subsequent protein nearby might be minimized if one domain could shuffle to a new site while the other two remain attached.

Multi-domain arrangements.tiff

Our final model for the association of PTB (black) while it represses the exon is shown below. Other proteins are likely to be involved.

PTB on TM

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