Advertisement

Accurate Mapping of Cleavage and Polyadenylation Sites by 3′ Region Extraction and Deep Sequencing

  • Mainul Hoque
  • Wencheng Li
  • Bin Tian
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1125)

Abstract

Deep sequencing of RNA (RNA-seq) is becoming a standard method to study gene expression. While RNA-seq reads cover most regions of an mRNA sequence, they are often depleted in the 3′ end region, making them less amenable for mapping the cleavage and polyadenylation site (pA). A major problem in identification of pA is mispriming at internal A-rich regions and oligo(A) tails when an oligo(dT) primer is used for reverse transcription or sequencing. We recently developed a method named 3′ region extraction and deep sequencing (3′READS), which completely addresses mispriming issues and is straightforward to use. The method accurately maps pAs and allows quantitative analysis of alternative cleavage and polyadenylation (APA) isoforms and gene expression.

Key words

Deep sequencing Gene expression Cleavage and polyadenylation PolyA site RNA-seq 

Notes

Acknowledgments

We thank other members of the BT lab for helpful comments and suggestions. This work was funded by an NIH grant (GM084089) to BT.

References

  1. 1.
    Edmonds M (2002) A history of poly A sequences: from formation to factors to function. Prog Nucleic Acid Res Mol Biol 71:285–389PubMedCrossRefGoogle Scholar
  2. 2.
    Colgan DF, Manley JL (1997) Mechanism and regulation of mRNA polyadenylation. Genes Dev 11:2755–2766PubMedCrossRefGoogle Scholar
  3. 3.
    Proudfoot NJ (2011) Ending the message: poly(A) signals then and now. Genes Dev 25:1770–1782PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Tian B, Graber JH (2012) Signals for pre-mRNA cleavage and polyadenylation. Wiley Interdiscip Rev RNA 3(3):385–396PubMedCrossRefGoogle Scholar
  5. 5.
    Tian B, Hu J, Zhang H et al (2005) A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33:201–212PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    di Giammartino DC, Nishida K, Manley JL (2011) Mechanisms and consequences of alternative polyadenylation. Mol Cell 43:853–866PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Lutz CS, Moreira A (2011) Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression. WIREs RNA 2:23–31PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Zhang H, Lee JY, Tian B (2005) Biased alternative polyadenylation in human tissues. Genome Biol 6:R100PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Wang ET, Sandberg R, Luo S et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Ji Z, Lee JY, Pan Z et al (2009) Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc Natl Acad Sci U S A 106:7028–7033PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Sandberg R, Neilson JR, Sarma A et al (2008) Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science 320:1643–1647PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Mayr C, Bartel DP (2009) Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138:673–684PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Singh P, Alley TL, Wright SM et al (2009) Global changes in processing of mRNA 3′ untranslated regions characterize clinically distinct cancer subtypes. Cancer Res 69:9422–9430PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Flavell SW, Kim TK, Gray JM et al (2008) Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection. Neuron 60:1022–1038PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Lee JY, Yeh I, Park JY et al (2007) PolyA_DB 2: mRNA polyadenylation sites in vertebrate genes. Nucleic Acids Res 35:D165–D168PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Brockman JM, Singh P, Liu D et al (2005) PACdb: polyA cleavage site and 3′-UTR database. Bioinformatics 21:3691–3693PubMedCrossRefGoogle Scholar
  17. 17.
    Nam DK, Lee S, Zhou G et al (2002) Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription. Proc Natl Acad Sci U S A 99:6152–6156PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wlotzka W, Kudla G, Granneman S et al (2011) The nuclear RNA polymerase II surveillance system targets polymerase III transcripts. EMBO J 30:1790–1803PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Schmidt MJ, Norbury CJ (2010) Polyadenylation and beyond: emerging roles for noncanonical poly(A) polymerases. Wiley Interdiscip Rev RNA 1:142–151PubMedGoogle Scholar
  20. 20.
    Hoque M, Ji Z, Zheng D et al (2013) Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing. Nat Methods 10:133–139PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Jayaprakash AD, Jabado O, Brown BD et al (2011) Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res 39:e141PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Zhuang F, Fuchs RT, Sun Z et al (2012) Structural bias in T4 RNA ligase-mediated 3′-adapter ligation. Nucleic Acids Res 40:e54PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mainul Hoque
    • 1
  • Wencheng Li
    • 1
  • Bin Tian
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical SchoolNewarkUSA

Personalised recommendations