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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

Published on: July 2, 2010

Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs

Markus Landthaler1, Dimos Gaidatzis, Andrea Rothballer

  • 1Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.

RNA (New York, N.Y.)
|November 4, 2008
PubMed

Insights

This study identifies trinucleotide repeat containing proteins 6 (TNRC6) as key components of microRNA (miRNA) silencing complexes. These findings reveal functional redundancy in human miRNA pathways and offer a new method for target identification.

Area of Science:

  • Molecular Biology
  • RNA Biology
  • Gene Regulation

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression, operating via miRNA-containing ribonucleoprotein particles (RNPs).
  • Argonaute (AGO) proteins are central to these complexes, binding miRNAs and facilitating target mRNA recognition.

Purpose of the Study:

  • To biochemically characterize the protein composition of human miRNA-containing RNPs.
  • To identify novel components and understand the functional roles of proteins within these silencing complexes.
  • To develop an effective approach for identifying physiologically relevant miRNA targets.

Main Methods:

  • Generation of HEK293 cell lines stably expressing epitope-tagged human AGO proteins and related RNA silencing factors.
  • Purification of miRNA-containing RNPs using affinity-based methods.
  • Mass spectrometry to analyze associated proteins and mRNA microarray analysis to identify bound transcripts.

Main Results:

  • Proteomic analysis revealed a common set of helicases and mRNA-binding proteins associated with different AGO proteins, including TNRC6A, TNRC6B, and TNRC6C.
  • AGO and TNRC6 proteins were found to bind highly similar sets of transcripts, which were enriched for binding sites of highly expressed endogenous miRNAs.
  • This indicates that TNRC6 proteins are integral components of the mRNA-targeting miRNA silencing complex.

Conclusions:

  • TNRC6 proteins are essential components of the miRNA-mediated gene silencing machinery.
  • The similar proteomic composition of AGO complexes suggests significant functional redundancy within human AGO and TNRC6 protein families.
  • The developed biochemical approach is effective for identifying physiologically relevant human miRNA targets.

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