Genome-Wide Probing of RNA Structures In Vitro Using Nucleases and Deep Sequencing

  • Yue Wan
  • Kun Qu
  • Zhengqing Ouyang
  • Howard Y. Chang
Part of the Methods in Molecular Biology book series (MIMB, volume 1361)

Abstract

RNA structure probing is an important technique that studies the secondary and tertiary conformations of an RNA. While it was traditionally performed on one RNA at a time, recent advances in deep sequencing has enabled the secondary structure mapping of thousands of RNAs simultaneously. Here, we describe the method Parallel Analysis for RNA Structures (PARS), which couples double and single strand specific nuclease probing to high throughput sequencing. Upon cloning of the cleavage sites into a cDNA library, deep sequencing and mapping of reads to the transcriptome, the position of paired and unpaired bases along cellular RNAs can be identified. PARS can be performed under diverse solution conditions and on different organismal RNAs to provide genome-wide RNA structural information. This information can also be further used to constrain computational predictions to provide better RNA structure models under different conditions.

Key words

RNA Structure Biochemistry Genomics High-throughput sequencing 

References

  1. 1.
    Wan Y, Kertesz M, Spitale RC, Segal E, Chang HY (2011) Understanding the transcriptome through RNA structure. Nat Rev Genet 12:641–655CrossRefPubMedGoogle Scholar
  2. 2.
    Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20:295–304PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Ehresmann C et al (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Mitra S, Shcherbakova IV, Altman RB, Brenowitz M, Laederach A (2008) High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res 36, e63PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Lucks JB et al (2011) Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A 108:11063–11068PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Underwood JG et al (2010) FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat Methods 7:995–1001PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Kertesz M et al (2010) Genome-wide measurement of RNA secondary structure in yeast. Nature 467:103–107CrossRefPubMedGoogle Scholar
  8. 8.
    Wan Y et al (2012) Genome-wide measurement of RNA folding energies. Mol Cell 48:169–181PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Wan Y et al (2014) Landscape and variation of RNA secondary structure across the human transcriptome. Nature 505:706–709PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Ouyang Z, Snyder MP, Chang H (2013) SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data. Genome Res 23(2):377–387PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Guo F, Gooding AR, Cech TR (2004) Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site. Mol Cell 16:351–362PubMedGoogle Scholar
  12. 12.
    Quail MA et al (2008) A large genome center’s improvements to the Illumina sequencing system. Nat Methods 5:1005–1010PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yue Wan
    • 1
  • Kun Qu
    • 2
  • Zhengqing Ouyang
    • 3
    • 4
  • Howard Y. Chang
    • 2
  1. 1.Stem Cell and Developmental BiologyGenome Institute of SingaporeSingaporeSingapore
  2. 2.Howard Hughes Medical Institute and Program in Epithelial BiologyStanford University School of MedicineStanfordUSA
  3. 3.The Jackson Laboratory for Genomic MedicineFarmingtonUSA
  4. 4.Department of Biomedical EngineeringUniversity of ConnecticutStorrsUSA

Personalised recommendations