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DNA Diagnostics and Exon Skipping

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Exon Skipping

Part of the book series: Methods in Molecular Biology ((MIMB,volume 867))

Abstract

The predominate form of DNA diagnostics remains nucleic acid sequencing in the research and clinical setting. While DNA sequencing allows a mutation to be correctly identified, only RNA sequencing can confirm the effect of that mutation on the resulting mRNA transcript. In the absence of RNA sequencing, predictions are reliant on either experimental studies or bioinformatic modelling. While each of these approaches provides insights into cellular splicing choices, of which exon skipping is but one, both possess inherent weaknesses. A method which is able to integrate and appropriately weigh the various factors influencing cellular splicing choices into an accurate, comprehensive modelling tool still remains elusive.

In this overview chapter, the current methods utilised for DNA diagnostics and the impact of the emerging next-generation sequencing techniques are considered. We explore why RNA remains a problematic medium with which to work. To understand how exon skipping can be predicted from a DNA sequence, the key cis-acting elements influencing splicing are reviewed. Finally, the current methods used to predict exon skipping including RNA-based studies, experimental studies, and bioinformatic modelling approaches are outlined.

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References

  1. Lopez-Bigas N, Audit B, Ouzounis C et al (2005) Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900–1903

    Article  PubMed  CAS  Google Scholar 

  2. Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564

    Article  PubMed  CAS  Google Scholar 

  3. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  4. Hyman ED (1988) A new method of sequencing DNA. Anal Biochem 174:423–436

    Article  PubMed  CAS  Google Scholar 

  5. Brenner S, Johnson M, Bridgham J et al (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 18:630–634

    Article  PubMed  CAS  Google Scholar 

  6. Shendure J, Porreca GJ, Reppas NB et al (2005) Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728–1732

    Article  PubMed  CAS  Google Scholar 

  7. Kasianowicz JJ, Brandin E, Branton D et al (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci USA 93:13770–13773

    Article  PubMed  CAS  Google Scholar 

  8. Dredge BK, Darnell RB (2003) Nova regulates GABA(A) receptor gamma2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer. Mol Cell Biol 23:4687–4700

    Article  PubMed  CAS  Google Scholar 

  9. Saenger W (1984) Principles of nucleic acid structure. Springer, New York

    Book  Google Scholar 

  10. Grabowski PJ, Zaug AJ, Cech TR (1981) The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena. Cell 23:467–476

    Article  PubMed  CAS  Google Scholar 

  11. Yang E, van Nimwegen E, Zavolan M et al (2003) Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes. Genome Res 13:1863–1872

    Article  PubMed  CAS  Google Scholar 

  12. Blank A, Gallant JA, Burgess RR et al (1986) An RNA polymerase mutant with reduced accuracy of chain elongation. Biochemistry 25:5920–5928

    Article  PubMed  CAS  Google Scholar 

  13. de Mercoyrol L, Corda Y, Job C et al (1992) Accuracy of wheat-germ RNA polymerase II. General enzymatic properties and effect of template conformational transition from right-handed B-DNA to left-handed Z-DNA. Eur J Biochem 206:49–58

    Article  PubMed  Google Scholar 

  14. Tourriere H, Chebli K, Tazi J (2002) mRNA degradation machines in eukaryotic cells. Biochimie 84:821–837

    Article  PubMed  CAS  Google Scholar 

  15. Fenger-Gron M, Fillman C, Norrild B et al (2005) Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol Cell 20:905–915

    Article  PubMed  CAS  Google Scholar 

  16. Xiang S, Cooper-Morgan A, Jiao X et al (2009) Structure and function of the 5′  ®  3′ exoribonuclease Rat1 and its activating partner Rai1. Nature 458:784–788

    Article  PubMed  CAS  Google Scholar 

  17. Dziembowski A, Lorentzen E, Conti E et al (2007) A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol Biol 14:15–22

    Article  PubMed  CAS  Google Scholar 

  18. Schneider C, Leung E, Brown J et al (2009) The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome. Nucleic Acids Res 37:1127–1140

    Article  PubMed  CAS  Google Scholar 

  19. Allmang C, Petfalski E, Podtelejnikov A et al (1999) The yeast exosome and human PM-Scl are related complexes of 3′  →  5′ exonucleases. Genes Dev 13:2148–2158

    Article  PubMed  CAS  Google Scholar 

  20. Jing Q, Huang S, Guth S et al (2005) Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120:623–634

    Article  PubMed  CAS  Google Scholar 

  21. Skruzny M, Schneider C, Racz A et al (2009) An endoribonuclease functionally linked to perinuclear mRNP quality control associates with the nuclear pore complexes. PLoS Biol 7:e8

    Article  PubMed  Google Scholar 

  22. Liu J, Carmell MA, Rivas FV et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441

    Article  PubMed  CAS  Google Scholar 

  23. Maquat LE (2005) Nonsense-mediated mRNA decay in mammals. J Cell Sci 118:1773–1776

    Article  PubMed  CAS  Google Scholar 

  24. Le Hir H, Izaurralde E, Maquat LE et al (2000) The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon-exon junctions. EMBO J 19:6860–6869

    Article  PubMed  Google Scholar 

  25. Vasudevan S, Peltz SW, Wilusz CJ (2002) Non-stop decay – a new mRNA surveillance pathway. Bioessays 24:785–788

    Article  PubMed  CAS  Google Scholar 

  26. Doma MK, Parker R (2006) Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440:561–564

    Article  PubMed  CAS  Google Scholar 

  27. Clark F, Thanaraj TA (2002) Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human. Hum Mol Genet 11:451–464

    Article  PubMed  CAS  Google Scholar 

  28. Dietz HC, Valle D, Francomano CA et al (1993) The skipping of constitutive exons in vivo induced by nonsense mutations. Science 259:680–683

    Article  PubMed  CAS  Google Scholar 

  29. Hentze MW, Kulozik AE (1999) A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96:307–310

    Article  PubMed  CAS  Google Scholar 

  30. Mendell JT, Dietz HC (2001) When the message goes awry: disease-producing mutations that influence mRNA content and performance. Cell 107:411–414

    Article  PubMed  CAS  Google Scholar 

  31. Nilsen TW, Graveley BR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463:457–463

    Article  PubMed  CAS  Google Scholar 

  32. Serra MJ, Turner DH (1995) Predicting thermodynamic properties of RNA. Methods Enzymol 259:242–261

    Article  PubMed  CAS  Google Scholar 

  33. Zhuang Y, Weiner AM (1986) A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation. Cell 46:827–835

    Article  PubMed  CAS  Google Scholar 

  34. Seraphin B, Kretzner L, Rosbash M (1988) A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J 7:2533–2538

    PubMed  CAS  Google Scholar 

  35. Siliciano PG, Guthrie C (1988) 5′ splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev 2:1258–1267

    Article  PubMed  CAS  Google Scholar 

  36. Freund M, Asang C, Kammler S et al (2003) A novel approach to describe a U1 snRNA binding site. Nucleic Acids Res 31:6963–6975

    Article  PubMed  CAS  Google Scholar 

  37. McCullough AJ, Berget SM (1997) G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection. Mol Cell Biol 17:4562–4571

    PubMed  CAS  Google Scholar 

  38. Del Gatto-Konczak F, Bourgeois CF, Le Guiner C et al (2000) The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5′ splice site. Mol Cell Biol 20:6287–6299

    Article  PubMed  Google Scholar 

  39. Hicks MJ, Mueller WF, Shepard PJ et al (2010) Competing upstream 5′ splice sites enhance the rate of proximal splicing. Mol Cell Biol 30:1878–1886

    Article  PubMed  CAS  Google Scholar 

  40. Zamore PD, Green MR (1989) Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci USA 86:9243–9247

    Article  PubMed  CAS  Google Scholar 

  41. Reed R, Maniatis T (1988) The role of the mammalian branchpoint sequence in pre-mRNA splicing. Genes Dev 2:1268–1276

    Article  PubMed  CAS  Google Scholar 

  42. Berglund JA, Chua K, Abovich N et al (1997) The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89:781–787

    Article  PubMed  CAS  Google Scholar 

  43. Liu HX, Zhang M, Krainer AR (1998) Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev 12:1998–2012

    Article  PubMed  CAS  Google Scholar 

  44. Fairbrother WG, Yeh RF, Sharp PA et al (2002) Predictive identification of exonic splicing enhancers in human genes. Science 297:1007–1013

    Article  PubMed  CAS  Google Scholar 

  45. Zhang XH, Chasin LA (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18:1241–1250

    Article  PubMed  CAS  Google Scholar 

  46. Wang Z, Rolish ME, Yeo G et al (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119:831–845

    Article  PubMed  CAS  Google Scholar 

  47. Del Gatto-Konczak F, Olive M, Gesnel MC et al (1999) hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol Cell Biol 19:251–260

    PubMed  Google Scholar 

  48. Wagner EJ, Garcia-Blanco MA (2001) Polypyrimidine tract binding protein antagonizes exon definition. Mol Cell Biol 21:3281–3288

    Article  PubMed  CAS  Google Scholar 

  49. Chen CD, Kobayashi R, Helfman DM (1999) 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

    Article  PubMed  CAS  Google Scholar 

  50. Chastain M, Tinoco I Jr (1991) Structural elements in RNA. Prog Nucleic Acid Res Mol Biol 41:131–177

    Article  PubMed  CAS  Google Scholar 

  51. Hermann T, Patel DJ (1999) Stitching together RNA tertiary architectures. J Mol Biol 294:829–849

    Article  PubMed  CAS  Google Scholar 

  52. Hiller M, Zhang Z, Backofen R et al (2007) Pre-mRNA secondary structures influence exon recognition. PLoS Genet 3:e204

    Article  PubMed  Google Scholar 

  53. Eperon LP, Graham IR, Griffiths AD et al (1988) 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

    Article  PubMed  CAS  Google Scholar 

  54. de la Mata M, Alonso CR, Kadener S et al (2003) A slow RNA polymerase II affects alternative splicing in vivo. Mol Cell 12:525–532

    Article  PubMed  Google Scholar 

  55. Lin S, Coutinho-Mansfield G, Wang D et al (2008) The splicing factor SC35 has an active role in transcriptional elongation. Nat Struct Mol Biol 15:819–826

    Article  PubMed  CAS  Google Scholar 

  56. Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–413

    Article  PubMed  CAS  Google Scholar 

  57. Rossi F, Labourier E, Forne T et al (1996) Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature 381:80–82

    Article  PubMed  CAS  Google Scholar 

  58. Adams MD, Kelley JM, Gocayne JD et al (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252:1651–1656

    Article  PubMed  CAS  Google Scholar 

  59. Johnson JM, Castle J, Garrett-Engele P et al (2003) Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302:2141–2144

    Article  PubMed  CAS  Google Scholar 

  60. Blanchette M, Green RE, Brenner SE et al (2005) Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 19:1306–1314

    Article  PubMed  CAS  Google Scholar 

  61. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  PubMed  CAS  Google Scholar 

  62. Wang ET, Sandberg R, Luo S et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476

    Article  PubMed  CAS  Google Scholar 

  63. Metherell LA, Akker SA, Munroe PB et al (2001) Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. Am J Hum Genet 69:641–646

    Article  PubMed  CAS  Google Scholar 

  64. Akker SA, Misra S, Aslam S et al (2007) Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon. Mol Endocrinol 21:2529–2540

    Article  PubMed  CAS  Google Scholar 

  65. Kornblihtt AR (2005) Promoter usage and alternative splicing. Curr Opin Cell Biol 17:262–268

    Article  PubMed  CAS  Google Scholar 

  66. Brunak S, Engelbrecht J, Knudsen S (1991) Prediction of human mRNA donor and acceptor sites from the DNA sequence. J Mol Biol 220:49–65

    Article  PubMed  CAS  Google Scholar 

  67. Vapnik V (1998) Statistical learning theory. Wiley-Interscience. ISBN 0-471-03003-1. New York

    Google Scholar 

  68. Staden R (1984) Computer methods to locate signals in nucleic acid sequences. Nucleic Acids Res 12:505–519

    Article  PubMed  CAS  Google Scholar 

  69. Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174

    Article  PubMed  CAS  Google Scholar 

  70. Salzberg SL (1997) A method for identifying splice sites and translational start sites in eukaryotic mRNA. Comput Appl Biosci 13:365–376

    PubMed  CAS  Google Scholar 

  71. Zhang MQ, Marr TG (1993) A weight array method for splicing signal analysis. Comput Appl Biosci 9:499–509

    PubMed  CAS  Google Scholar 

  72. Burge C, Karlin S (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268:78–94

    Article  PubMed  CAS  Google Scholar 

  73. Smith PJ, Zhang C, Wang J et al (2006) An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet 15:2490–2508

    Article  PubMed  CAS  Google Scholar 

  74. Cartegni L, Wang J, Zhu Z et al (2003) ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res 31:3568–3571

    Article  PubMed  CAS  Google Scholar 

  75. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510

    Article  PubMed  CAS  Google Scholar 

  76. Eddy SR (2004) What is a hidden Markov model? Nat Biotechnol 22:1315–1316

    Article  PubMed  CAS  Google Scholar 

  77. Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11:377–394

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. St Bartholomew’s Hospital, London, UK

    Umasuthan Srirangalingam & Shern L. Chew

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  1. Umasuthan Srirangalingam
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  2. Shern L. Chew
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Correspondence to Umasuthan Srirangalingam .

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Editors and Affiliations

  1. Dept. Human & Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, Leiden, 2333 ZC, Netherlands

    Annemieke Aartsma-Rus

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Srirangalingam, U., Chew, S.L. (2012). DNA Diagnostics and Exon Skipping. In: Aartsma-Rus, A. (eds) Exon Skipping. Methods in Molecular Biology, vol 867. Humana Press. https://doi.org/10.1007/978-1-61779-767-5_1

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  • DOI: https://doi.org/10.1007/978-1-61779-767-5_1

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-766-8

  • Online ISBN: 978-1-61779-767-5

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