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PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
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Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

Rongbing Huang1,2, Mengting Han1,2, Liying Meng1,3

  • 1College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, China.

Proceedings of the National Academy of Sciences of the United States of America
|April 12, 2018
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method to identify all RNA-binding proteins (RBPs), including those on noncoding RNAs. This discovery expands our understanding of gene regulation and reveals new roles for noncoding RNAs.

Keywords:
RNARNA–protein interactionsbioorthogonal chemistrynoncoding RNAproteomics

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Area of Science:

  • Molecular Biology
  • Proteomics
  • Genomics

Background:

  • Understanding RNA-binding proteins (RBPs) is crucial for deciphering gene regulation.
  • Current methods primarily identify RBPs binding to polyadenylated (poly(A)) mRNA, neglecting RBPs associated with nonpoly(A) RNAs like noncoding RNAs (ncRNAs) and pre-mRNAs.

Purpose of the Study:

  • To develop an unbiased method for identifying all RBPs, regardless of their RNA target's polyadenylation status.
  • To expand the known repertoire of RBPs and explore their potential roles in ncRNA-mediated gene regulation.

Main Methods:

  • Developed the click chemistry-assisted RNA interactome capture (CARIC) strategy.
  • Combined metabolic RNA labeling with an alkynyl uridine analog, in vivo photocross-linking, and click chemistry with azide-biotin.
  • Utilized affinity enrichment and proteomic analysis to identify bound proteins.

Main Results:

  • Identified 597 RBPs in HeLa cells using the CARIC method.
  • Discovered 130 novel RBPs, expanding the known RBP landscape.
  • These novel RBPs are potential binders of ncRNAs, suggesting new regulatory functions.

Conclusions:

  • The CARIC strategy provides an unbiased approach for comprehensive RBP identification.
  • Newly identified RBPs may mediate ncRNA functions in diverse cellular processes, including proteasome function and metabolism.
  • CARIC is applicable across species, facilitating a complete census of RBPs and their regulatory roles.