Advertisement

Using Yeast Genetics to Study Splicing Mechanisms

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1126)

Abstract

Pre-mRNA splicing is a critical step in eukaryotic gene expression, which involves removal of noncoding intron sequences from pre-mRNA and ligation of the remaining exon sequences to make a mature message. Splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Since the first description of the pre-mRNA splicing reaction in the 1970s, elegant genetic and biochemical studies have revealed that the enzyme that catalyzes the reaction, the spliceosome, is an exquisitely dynamic macromolecular machine, and its RNA and protein components undergo highly ordered, tightly coordinated rearrangements in order to carry out intron recognition and splicing catalysis. Studies using the genetically tractable unicellular eukaryote budding yeast (Saccharomyces cerevisiae) have played an instrumental role in deciphering splicing mechanisms. In this chapter, we discuss how yeast genetics has been used to deepen our understanding of the mechanism of splicing and explore the potential for future mechanistic insights using S. cerevisiae as an experimental tool.

Key words

Pre-mRNA Splicing Saccharomyces cerevisiae Yeast genetics Synthetic lethality Temperature-sensitive (ts) screening Suppressor screening DExD/H-box protein SGA analysis E-MAP 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We would like to thank Drs. Kristin Patrick (UCSF) and Lorraine Pillus (UCSD) for critical reading of the manuscript. Funding was provided by the National Institutes of General Medical Sciences (GM085764) and the National Science Foundation (MCB-1051921).

References

  1. 1.
    Jurica MS, Moore MJ (2003) Pre-mRNA splicing: awash in a sea of proteins. Mol Cell 12(1):5–14PubMedCrossRefGoogle Scholar
  2. 2.
    Wahl MC, Will CL, Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136(4):701–718PubMedCrossRefGoogle Scholar
  3. 3.
    Ares M Jr, Grate L, Pauling MH (1999) A handful of intron-containing genes produces the lion’s share of yeast mRNA. RNA 5(9):1138–1139PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Sherman F (1997) An introduction to the genetics and molecular biology of the yeast Saccharomyces cerevisiae. In: Meyers RA (ed) The encyclopedia of molecular biology and molecular medicine. VCH Publisher, Weinheim, Germany, pp 302–325Google Scholar
  5. 5.
    Hartwell LH (1967) Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol 93(5):1662–1670PubMedCentralPubMedGoogle Scholar
  6. 6.
    Hartwell LH, McLaughlin CS, Warner JR (1970) Identification of ten genes that control ribosome formation in yeast. Mol Gen Genet 109(1):42–56PubMedCrossRefGoogle Scholar
  7. 7.
    Rosbash M, Harris PK, Woolford JL Jr et al (1981) The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell 24(3):679–686PubMedCrossRefGoogle Scholar
  8. 8.
    Lustig AJ, Lin RJ, Abelson J (1986) The yeast RNA gene products are essential for mRNA splicing in vitro. Cell 47(6):953–963PubMedCrossRefGoogle Scholar
  9. 9.
    Vijayraghavan U, Company M, Abelson J (1989) Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev 3(8):1206–1216PubMedCrossRefGoogle Scholar
  10. 10.
    Noble SM, Guthrie C (1996) Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations. Genetics 143(1):67–80PubMedCentralPubMedGoogle Scholar
  11. 11.
    Brachmann CB, Davies A, Cost GJ et al (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2):115–132PubMedCrossRefGoogle Scholar
  12. 12.
    Hawley RS, Walker MY (2003) Advanced genetic analysis: finding meaning in a genome. Blackwell Publishing, Malden, MAGoogle Scholar
  13. 13.
    Parker R, Guthrie C (1985) A point mutation in the conserved hexanucleotide at a yeast 5′ splice junction uncouples recognition, cleavage, and ligation. Cell 41(1):107–118PubMedCrossRefGoogle Scholar
  14. 14.
    Vijayraghavan U, Parker R, Tamm J et al (1986) Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J 5(7):1683–1695PubMedCentralPubMedGoogle Scholar
  15. 15.
    Couto JR, Tamm J, Parker R et al (1987) A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes Dev 1(5):445–455PubMedCrossRefGoogle Scholar
  16. 16.
    Parker R, Siliciano PG, Guthrie C (1987) Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49(2):229–239PubMedCrossRefGoogle Scholar
  17. 17.
    Siliciano PG, Guthrie C (1988) 5′ splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev 2(10):1258–1267PubMedCrossRefGoogle Scholar
  18. 18.
    Schwer B, Guthrie C (1991) PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature 349(6309):494–499PubMedCrossRefGoogle Scholar
  19. 19.
    Burgess SM, Guthrie C (1993) A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell 73(7):1377–1391PubMedCrossRefGoogle Scholar
  20. 20.
    Chang TH, Tung L, Yeh FL et al (2013) Functions of the DExD/H-box proteins in nuclear pre-mRNA splicing. Biochim Biophys Acta 1829(8):764–774PubMedCrossRefGoogle Scholar
  21. 21.
    Villa T, Guthrie C (2005) The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev 19(16):1894–1904PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Semlow DR, Staley JP (2012) Staying on message: ensuring fidelity in pre-mRNA splicing. Trends Biochem Sci 37(7):263–273PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Koodathingal P, Staley JP (2013) Splicing fidelity: DEAD/H-box ATPases as molecular clocks. RNA Biol 10(7)Google Scholar
  24. 24.
    Winzeler EA, Shoemaker DD, Astromoff A et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429):901–906PubMedCrossRefGoogle Scholar
  25. 25.
    Hartman JL, Garvik B, Hartwell L (2001) Principles for the buffering of genetic variation. Science 291(5506):1001–1004PubMedCrossRefGoogle Scholar
  26. 26.
    Boone C, Bussey H, Andrews BJ (2007) Exploring genetic interactions and networks with yeast. Nat Rev Genet 8(6):437–449PubMedCrossRefGoogle Scholar
  27. 27.
    Collins SR, Roguev A, Krogan NJ (2010) Quantitative genetic interaction mapping using the E-MAP approach. Methods Enzymol 470:205–231PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Perriman R, Ares M Jr (2000) ATP can be dispensable for prespliceosome formation in yeast. Genes Dev 14(1):97–107PubMedCentralPubMedGoogle Scholar
  29. 29.
    Perriman R, Barta I, Voeltz GK et al (2003) ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA. Proc Natl Acad Sci USA 100(24):13857–13862PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Hossain MA, Claggett JM, Nguyen T et al (2009) The cap binding complex influences H2B ubiquitination by facilitating splicing of the SUS1 pre-mRNA. RNA 15(8):1515–1527PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Breslow DK, Cameron DM, Collins SR et al (2008) A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods 5(8):711–718PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Tong AH, Boone C (2006) Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol Biol 313:171–192PubMedGoogle Scholar
  33. 33.
    Baryshnikova A, Costanzo M, Dixon S et al (2010) Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Methods Enzymol 470:145–179PubMedCrossRefGoogle Scholar
  34. 34.
    Tong AH, Evangelista M, Parsons AB et al (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294(5550):2364–2368PubMedCrossRefGoogle Scholar
  35. 35.
    Tong AH, Lesage G, Bader GD et al (2004) Global mapping of the yeast genetic interaction network. Science 303(5659):808–813PubMedCrossRefGoogle Scholar
  36. 36.
    Baryshnikova A, Costanzo M, Kim Y et al (2010) Quantitative analysis of fitness and genetic interactions in yeast on a genome scale. Nat Methods 7(12):1017–1024PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Fiedler D, Braberg H, Mehta M et al (2009) Functional organization of the S. cerevisiae phosphorylation network. Cell 136(5):952–963PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Schuldiner M, Collins SR, Thompson NJ et al (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123(3):507–519PubMedCrossRefGoogle Scholar
  39. 39.
    Wilmes GM, Bergkessel M, Bandyopadhyay S et al (2008) A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing. Mol Cell 32(5):735–746PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Kress TL, Krogan NJ, Guthrie C (2008) A single SR-like protein, Npl3, promotes pre-mRNA splicing in budding yeast. Mol Cell 32(5):727–734PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Moehle EA, Ryan CJ, Krogan NJ et al (2012) The yeast SR-like protein Npl3 links chromatin modification to mRNA processing. PLoS Genet 8(11):e1003101PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Gunderson FQ, Johnson TL (2009) Acetylation by the transcriptional coactivator Gcn5 plays a novel role in co-transcriptional spliceosome assembly. PLoS Genet 5(10):e1000682PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Gunderson FQ, Merkhofer EC, Johnson TL (2011) Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements. Proc Natl Acad Sci USA 108(5):2004–2009PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Hossain MA, Chung C, Pradhan SK et al (2013) The yeast cap binding complex modulates transcription factor recruitment and establishes proper histone H3K36 trimethylation during active transcription. Mol Cell Biol 33(4):785–799PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  1. 1.Molecular Biology Section, Division of Biological SciencesUniversity of California, San DiegoLa JollaUSA
  2. 2.Department of Molecular, Cell and Developmental BiologyUniversity of California, Los AngelesLos AngelesUSA

Personalised recommendations

Cite protocol

Buy options