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Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq.

Publication ,  Journal Article
Nicholson, CO; Friedersdorf, M; Keene, JD
Published in: RNA
January 2017

RNA-binding proteins (RBPs) and noncoding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site and quantifies binding to whole transcripts. In this study, we compare current procedures and present digestion optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. In addition, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole-transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets and binding sites where combinatorial interactions between HuR and AGO-microRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.

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Published In

RNA

DOI

EISSN

1469-9001

Publication Date

January 2017

Volume

23

Issue

1

Start / End Page

32 / 46

Location

United States

Related Subject Headings

  • Sequence Analysis, RNA
  • RNA, Messenger
  • Protein Binding
  • MicroRNAs
  • Humans
  • High-Throughput Nucleotide Sequencing
  • HEK293 Cells
  • Gene Expression Regulation
  • Gene Expression Profiling
  • ELAV-Like Protein 1
 

Citation

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Nicholson, C. O., Friedersdorf, M., & Keene, J. D. (2017). Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq. RNA, 23(1), 32–46. https://doi.org/10.1261/rna.058115.116
Nicholson, Cindo O., Matthew Friedersdorf, and Jack D. Keene. “Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq.RNA 23, no. 1 (January 2017): 32–46. https://doi.org/10.1261/rna.058115.116.
Nicholson CO, Friedersdorf M, Keene JD. Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq. RNA. 2017 Jan;23(1):32–46.
Nicholson, Cindo O., et al. “Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq.RNA, vol. 23, no. 1, Jan. 2017, pp. 32–46. Pubmed, doi:10.1261/rna.058115.116.
Nicholson CO, Friedersdorf M, Keene JD. Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq. RNA. 2017 Jan;23(1):32–46.

Published In

RNA

DOI

EISSN

1469-9001

Publication Date

January 2017

Volume

23

Issue

1

Start / End Page

32 / 46

Location

United States

Related Subject Headings

  • Sequence Analysis, RNA
  • RNA, Messenger
  • Protein Binding
  • MicroRNAs
  • Humans
  • High-Throughput Nucleotide Sequencing
  • HEK293 Cells
  • Gene Expression Regulation
  • Gene Expression Profiling
  • ELAV-Like Protein 1