High-Throughput Nuclease-Mediated Probing of RNA Secondary Structure in Plant Transcriptomes

  • Lee E. Vandivier
  • Fan Li
  • Brian D. GregoryEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1284)


Empirical measurement of RNA secondary structure is an invaluable tool that has provided a more complete understanding of the RNA life cycle and functionality of this extremely important molecule. In general, methods for probing structural information involve treating RNA with either a chemical or an enzyme that preferentially targets regions of the RNA in a single- or double-stranded conformation (ssRNA and dsRNA, respectively). Here, we describe an approach that utilizes a combination of ssRNA- and dsRNA-specific nuclease (ss- and dsRNase, respectively) treatments along with high-throughput sequencing technology to provide comprehensive and robust measurements of RNA secondary structure across entire plant transcriptomes.

Key words

RNA genomics RNA secondary structure Posttranscriptional regulation Transcriptome Nuclease probing 



RNA binding protein


Ribosomal RNA


Transfer RNA


Small nucleolar RNA


Long non-coding RNA


Single-stranded RNA


Double-stranded RNA


Single-stranded RNA nuclease


Double-stranded RNA nuclease


Polymerase chain reaction


Base pair


Small RNA


Small RNA sequencing


  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.
    Wanrooij PH, Uhler JP, Simonsson T, Falkenberg M, Gustafsson CM (2010) G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation. Proc Natl Acad Sci U S A 107:16072–16077CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Dong H et al (2007) Distinct RNA elements confer specificity to flavivirus RNA cap methylation events. J Virol 81:4412–4421CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Raker VA, Mironov AA, Gelfand MS, Pervouchine DD (2009) Modulation of alternative splicing by long-range RNA structures in Drosophila. Nucleic Acids Res 37:4533–4544CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Warf MB, Berglund JA (2010) The role of RNA structure in regulating pre-mRNA splicing. Trends Biochem Sci 35:169–178CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Oikawa D, Tokuda M, Hosoda A, Iwawaki T (2010) Identification of a consensus element recognized and cleaved by IRE1α. Nucleic Acids Res 38:6265–6273CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Klasens BIF, Das AT, Berkhout B (1998) Inhibition of polyadenylation by stable RNA secondary structure. Nucleic Acids Res 26:1870–1876CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Grüter P et al (1998) TAP, the Human Homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell 1:649–659CrossRefPubMedGoogle Scholar
  9. 9.
    Bullock SL, Ringel I, Ish-Horowicz D, Lukavsky PJ (2010) A′-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat Struct Mol Biol 17:703–709CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Subramanian M et al (2011) G–quadruplex RNA structure as a signal for neurite mRNA targeting. EMBO Rep 12:697–704CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Kozak M (1988) Leader length and secondary structure modulate mRNA function under conditions of stress. Mol Cell Biol 8:2737–2744PubMedCentralPubMedGoogle Scholar
  12. 12.
    Wen J-D et al (2008) Following translation by single ribosomes one codon at a time. Nature 452:598–603CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Svitkin YV et al (2001) The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5′ secondary structure. RNA 7:382–394CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Goodarzi H et al (2012) Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Nature 485:264–268CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Tsai M-C et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Kertesz M et al (2010) Genome-wide measurement of RNA secondary structure in yeast. Nature 467:103–107CrossRefPubMedGoogle Scholar
  17. 17.
    Li F et al (2012) Regulatory impact of RNA secondary structure across the arabidopsis transcriptome. Plant Cell 24:4346–4359CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Buratti E, Baralle FE (2004) Influence of RNA secondary structure on the pre-mRNA splicing process. Mol Cell Biol 24:10505–10514CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Trappl K, Polacek N (2011) The ribosome: a molecular machine powered by RNA. Met Ions Life Sci 9:253–275CrossRefPubMedGoogle Scholar
  20. 20.
    Schroeder R, Barta A, Semrad K (2004) Strategies for RNA folding and assembly. Nat Rev Mol Cell Biol 5:908–919CrossRefPubMedGoogle Scholar
  21. 21.
    Khalil AM, Rinn JL (2011) RNA–protein interactions in human health and disease. Semin Cell Dev Biol 22:359–365CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Rouskin S, Zubradt M, Washietl S, Kellis M, Weissman JS (2014) Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505:701–705CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Ding Y et al (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505:696–700CrossRefPubMedGoogle Scholar
  24. 24.
    Wilkinson KA, Merino EJ, Weeks KM (2006) Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610–1616CrossRefPubMedGoogle Scholar
  25. 25.
    Ehresmann C et al (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Siegfried NA, Busan S, Rice GM, Nelson JAE, Weeks KM (2014) RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods 11:959–965CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Qu X et al (2011) The ribosome uses two active mechanisms to unwind messenger RNA during translation. Nature 475:118–121CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Talkish J, May G, Lin Y, Woolford JL, McManus CJ (2014) Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA 20:713–720CrossRefPubMedGoogle Scholar
  29. 29.
    Li F et al (2012) Global analysis of RNA secondary structure in two metazoans. Cell Rep 1:69–82CrossRefPubMedGoogle Scholar
  30. 30.
    Mahalakshmi YV, Jagannadham MV, Pandit MW (2000) Ribonuclease from cobra snake venom: purification by affinity chromatography and further characterization. IUBMB Life 49:309–316CrossRefPubMedGoogle Scholar
  31. 31.
    Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12CrossRefGoogle Scholar
  32. 32.
    Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Kim D et al (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL (2008) The Vienna RNA Websuite. Nucleic Acids Res 36:W70–W74CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Lee E. Vandivier
    • 1
    • 2
    • 3
  • Fan Li
    • 1
    • 2
    • 4
  • Brian D. Gregory
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of BiologyUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.PENN Genome Frontiers InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Cell and Molecular Biology Graduate ProgramUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Genomics and Computational Biology Graduate ProgramUniversity of PennsylvaniaPhiladelphiaUSA

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