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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Related Experiment Video

Updated: Mar 13, 2026

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
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PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

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

Cindo O Nicholson1, Matthew Friedersdorf1, Jack D Keene1

  • 1Department of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

RNA (New York, N.Y.)
|October 16, 2016
PubMed
Summary
This summary is machine-generated.

Digestion optimized RIP-seq (DO-RIP-seq) quantifies RNA-binding protein (RBP) binding sites and whole transcripts, offering a continuous metric for RNA regulation. This method reveals RBP binding strength and functional targets, advancing post-transcriptional regulation studies.

Keywords:
CLIPRNA regulonsbinding site saturationcrosslinkprotein–RNA affinity

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • RNA-binding proteins (RBPs) and noncoding RNAs regulate gene expression post-transcriptionally.
  • Current methods for identifying RBP binding sites and targets have limitations in simultaneously measuring binding strength and whole-transcript regulation.
  • Understanding the precise relationship between RBP binding site affinity and transcript-level regulation is crucial.

Purpose of the Study:

  • To develop and validate a novel method, digestion optimized RIP-seq (DO-RIP-seq), for quantifying RBP binding sites and whole transcripts.
  • To compare the performance of DO-RIP-seq with existing techniques for RBP-RNA interaction analysis.
  • To investigate the role of the RBP HuR/ELAVL1 in post-transcriptional regulation using the new method.

Main Methods:

  • Development of digestion optimized RIP-seq (DO-RIP-seq), a method utilizing a continuous metric to locate and quantify RBP binding sites.
  • Application of DO-RIP-seq to the RBP HuR/ELAVL1 in human cells.
  • Validation of DO-RIP-seq by correlating quantitative binding site enrichment with in vitro binding strength.

Main Results:

  • DO-RIP-seq successfully quantified HuR binding sites across the human transcriptome with high coverage, providing metrics of relative RNA binding strength.
  • The quantitative enrichment of binding sites measured by DO-RIP-seq was proportional to their in vitro binding strength.
  • DO-RIP-seq enabled the quantification and comparison of HuR binding to whole transcripts, integrating binding site data with transcript-level measurements.
  • The method identified functional mRNA targets and binding sites involved in combinatorial regulation with AGO-microRNAs.

Conclusions:

  • DO-RIP-seq is a simple and effective method for locating and quantifying RBP binding sites with a continuous metric.
  • This technique allows for the simultaneous measurement of RBP binding site strength and whole-transcript binding.
  • DO-RIP-seq facilitates the identification of functional mRNA targets and provides insights into RBP-mediated regulation of dynamic biological processes.