Detection and Characterization of Transcription Termination

  • Ghada Ghazal
  • Jules Gagnon
  • Sherif Abou ElelaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


In most eukaryotes, the generation of the 3′ end and transcription termination are initiated by cleavage of the pre-mRNA upstream of the polyadenylation site. This cleavage initiates 5′–3′ degradation of the 3′ end cleavage product by the exoribonuclease Rat1p leading to the dissociation of the RNA polymerase II (RNAPII) complex. The Rat1p-dependent transcription termination was also shown to be initiated by a polyadenylation-independent cleavage performed by the double-stranded RNA-specific ribonuclease (RNase) III (Rnt1p) suggesting that the majority of transcription termination events are RNase dependent. Therefore, it became essential for future studies on transcription termination to carefully consider both the nature of the RNase-dependent RNA transcripts and the association pattern of the RNAPII with the transcriptional unit. Here, we present methods allowing the evaluation of the impact of yeast RNases on the 3′ end formation and their contribution to transcription termination. Northern blot analysis of transcripts generated downstream of known genes in the absence of RNases identifies potential transcription termination sites while chromatin immunoprecipitation of RNAPII differentiates between termination- and transcription-independent processing events.

Key words

RNase III Rat1p Rnt1p Termination ChIP Northern blot Ribonucleases 



This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institute of Health Research (CIHR).


  1. 1.
    Prescott EM, Osheim YN, Jones HS, et al (2004) Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p. Proc Natl Acad Sci USA 101:6068–73PubMedCrossRefGoogle Scholar
  2. 2.
    Cui M, Allen MA, Larsen A, Macmorris M, Han M, Blumenthal T (2008) Genes involved in pre-mRNA 3′-end formation and transcription termination revealed by a lin-15 operon Muv suppressor screen. Proc Natl Acad Sci USA 105:16665–70PubMedCrossRefGoogle Scholar
  3. 3.
    Zarudnaya MI, Kolomiets IM, Hovorun DM (2002) What nuclease cleaves pre-mRNA in the process of polyadenylation? IUBMB Life 54:27–31PubMedCrossRefGoogle Scholar
  4. 4.
    Tollervey D (2004) Molecular biology: termination by torpedo. Nature 432:456–7PubMedCrossRefGoogle Scholar
  5. 5.
    Luo W, Bentley D. (2004) A ribonucleolytic rat torpedoes RNA polymerase II. Cell 119:911–4PubMedCrossRefGoogle Scholar
  6. 6.
    West S, Gromak N, Proudfoot NJ (2004) Human 5′→3′ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 432:522–5PubMedCrossRefGoogle Scholar
  7. 7.
    Kim M, Vasiljeva L, Rando OJ, Zhelkovsky A, Moore C, Buratowski S (2006) Distinct pathways for snoRNA and mRNA termination. Mol Cell 24:723–34PubMedCrossRefGoogle Scholar
  8. 8.
    Carroll KL, Pradhan DA, Granek JA, Clarke ND, Corden JL (2004) Identification of cis elements directing termination of yeast nonpolyadenylated snoRNA transcripts. Mol Cell Biol 24:6241–52PubMedCrossRefGoogle Scholar
  9. 9.
    Osheim YN, French SL, Keck KM, et al (2004) Pre-18 S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae. Mol Cell 16:943–54PubMedCrossRefGoogle Scholar
  10. 10.
    Steinmetz EJ, Warren CL, Kuehner JN, Panbehi B, Ansari AZ, Brow DA (2006) Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase. Mol Cell 24:735–46PubMedCrossRefGoogle Scholar
  11. 11.
    Egloff S, O’Reilly D, Murphy S (2008) Expression of human snRNA genes from beginning to end. Biochem Soc Trans 36:590–4PubMedCrossRefGoogle Scholar
  12. 12.
    Richard P, Manley JL (2009) Transcription termination by nuclear RNA polymerases. Genes Dev 23:1247–69PubMedCrossRefGoogle Scholar
  13. 13.
    Ghazal G, Gagnon J, Jacques PE, Landry JR, Robert F, Elela SA (2009) Yeast RNase III triggers polyadenylation-independent transcription termination. Mol Cell 36:99–109PubMedCrossRefGoogle Scholar
  14. 14.
    Rondon AG, Mischo HE, Kawauchi J, Proudfoot NJ (2009) Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae. Mol Cell 36:88–98PubMedCrossRefGoogle Scholar
  15. 15.
    Taggart AK, Teng SC, Zakian VA (2002) Est1p as a cell cycle-regulated activator of telomere-bound telomerase. Science 297:1023–6PubMedCrossRefGoogle Scholar
  16. 16.
    Lamontagne B, Tremblay A, Abou Elela S (2000) The N-terminal domain that distinguishes yeast from bacterial RNase III contains a dimerization signal required for efficient double-stranded RNA cleavage. Mol Cell Biol 20:1104–15PubMedCrossRefGoogle Scholar
  17. 17.
    David L, Huber W, Granovskaia M, et al (2006) A high-resolution map of transcription in the yeast genome. Proc Natl Acad Sci U S A 103:5320–5PubMedCrossRefGoogle Scholar
  18. 18.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–86PubMedGoogle Scholar
  19. 19.
    SantaLucia J, Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–5PubMedCrossRefGoogle Scholar
  20. 20.
    Brosseau JP, Lucier JF, Lapointe E, et al (2010) High-throughput quantification of splicing isoforms. RNA 16:442–9PubMedCrossRefGoogle Scholar
  21. 21.
    Lopez R, Silventoinen V, Robinson S, Kibria A, Gish W (2003) WU-Blast2 server at the European Bioinformatics Institute. Nucleic Acids Res 31:3795–8PubMedCrossRefGoogle Scholar
  22. 22.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–5PubMedCrossRefGoogle Scholar
  23. 23.
    Catala M, Tremblay M, Samson E, Conconi A, Abou Elela S (2008) Deletion of Rnt1p alters the proportion of open versus closed rRNA gene repeats in yeast. Mol Cell Biol 28:619–29PubMedCrossRefGoogle Scholar
  24. 24.
    Cherry JM, Ball C, Weng S, et al (1997) Genetic and physical maps of Saccharomyces cerevisiae. Nature 387:67–73PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ghada Ghazal
    • 1
  • Jules Gagnon
    • 1
  • Sherif Abou Elela
    • 1
    Email author
  1. 1.RNA Group/Groupe ARN, Département de Microbiologie & Infectiologie, Faculté de médecine et des sciences de la santéUniversité de SherbrookeSherbrookeCanada

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