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Plant Functional Genomics pp 41–70Cite as

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

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1284))

Abstract

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.

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Abbreviations

RBP:

RNA binding protein

rRNA:

Ribosomal RNA

tRNA:

Transfer RNA

snoRNA:

Small nucleolar RNA

lncRNA:

Long non-coding RNA

ssRNA:

Single-stranded RNA

dsRNA:

Double-stranded RNA

ssRNase:

Single-stranded RNA nuclease

dsRNase:

Double-stranded RNA nuclease

PCR:

Polymerase chain reaction

bp:

Base pair

smRNA:

Small RNA

smRNA-seq:

Small RNA sequencing

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Author information

Authors and Affiliations

  1. Department of Biology, University of Pennsylvania, 433 S. University Ave., Philadelphia, PA, 19104, USA

    Lee E. Vandivier, Fan Li & Brian D. Gregory

  2. PENN Genome Frontiers Institute, University of Pennsylvania, Philadelphia, PA, USA

    Lee E. Vandivier, Fan Li & Brian D. Gregory

  3. Cell and Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, PA, USA

    Lee E. Vandivier & Brian D. Gregory

  4. Genomics and Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Fan Li & Brian D. Gregory

Authors
  1. Lee E. Vandivier
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  2. Fan Li
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  3. Brian D. Gregory
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Corresponding author

Correspondence to Brian D. Gregory .

Editor information

Editors and Affiliations

  1. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Jose M. Alonso

  2. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Anna N. Stepanova

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Vandivier, L.E., Li, F., Gregory, B.D. (2015). High-Throughput Nuclease-Mediated Probing of RNA Secondary Structure in Plant Transcriptomes. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-2444-8_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2443-1

  • Online ISBN: 978-1-4939-2444-8

  • eBook Packages: Springer Protocols

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