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Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification
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A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex

Joann Wu1, Zachary T Campbell, Elena Menichelli

  • 1Department of Molecular Biology, Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Journal of Molecular Biology
|November 20, 2012
PubMed

Insights

The fem-3 binding factor (FBF) and germline development defective-3 (GLD-3) interaction in C. elegans is key for spermatogenesis. Specific conserved regions in FBF and GLD-3 mediate this interaction, potentially forming a ternary complex with fem-3 mRNA.

Area of Science:

  • Molecular Biology
  • Developmental Biology
  • Genetics

Background:

  • Pumilio and FBF (PUF) proteins regulate mRNA post-transcriptionally.
  • The FBF and GLD-3 interaction is crucial for Caenorhabditis elegans spermatogenesis.
  • This interaction enhances fem-3 mRNA expression.

Purpose of the Study:

  • To elucidate the molecular basis of the FBF-GLD-3 interaction.
  • To identify the specific protein regions involved in binding.
  • To investigate the formation of complexes on the fem-3 mRNA.

Main Methods:

  • Protein interaction studies.
  • Identification of minimal binding regions within FBF and GLD-3.
  • Ternary complex formation assays.

Main Results:

  • A 10-amino acid region in GLD-3 and a 7-amino acid loop in FBF are essential for binding.
  • These binding sequences are conserved across related proteins.
  • GLD-3 and FBF form a ternary complex on the fem-3 mRNA's point mutation element without affecting FBF's RNA binding.

Conclusions:

  • The study defines the molecular interactions between FBF and GLD-3.
  • Conserved binding interfaces suggest functional importance.
  • A GLD-3⋅FBF⋅RNA complex is proposed as a regulatory mechanism for germline mRNAs.

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