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Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket

Chen Qiu1, Aaron Kershner, Yeming Wang

  • 1Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

The Journal of Biological Chemistry
|December 30, 2011
PubMed

Insights

Pumilio (PUF) proteins bind specific RNA sequences. A key adaptation, an upstream cytosine binding pocket, enhances RNA specificity and is found in diverse PUF proteins, contributing to regulatory network diversity.

Area of Science:

  • Molecular Biology
  • RNA Biology
  • Protein-RNA Interactions

Background:

  • mRNA control networks rely on specific RNA sequence recognition by RNA-binding proteins.
  • Pumilio-fem-3 mRNA binding factor (PUF) proteins utilize a conserved scaffold for RNA specificity.
  • Saccharomyces cerevisiae Puf3p employs an additional binding pocket for an upstream cytosine base.

Purpose of the Study:

  • To investigate the prevalence and functional significance of the upstream cytosine binding pocket in PUF proteins.
  • To understand how this structural adaptation contributes to the diversity of RNA specificities among PUF proteins.
  • To explore the evolutionary implications of the presence or absence of this binding pocket.

Main Methods:

  • Bioinformatics analysis of mRNA targets associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2.
  • Determination of crystal structures for FBF-2 and C. elegans PUF-6.
  • Sequence alignments to predict binding pockets in other PUF proteins, including PUF-11.
  • Experimental validation of the requirement for upstream pockets and cytosine for RNA binding.
  • Structure-based sequence alignments to identify key residues and evolutionary changes.

Main Results:

  • The upstream cytosine binding pocket is a prevalent adaptation among PUF proteins, including FBF-2, PUF-6, and PUF-11.
  • This pocket and an upstream cytosine are necessary for maximal RNA binding in Puf3p, FBF-2, PUF-6, and PUF-11, though binding affinity impacts vary.
  • The position of the upstream cytosine can vary, and some PUF proteins lack this pocket due to key residue differences or interfering amino acids.
  • Loss of the pocket can occur through a single serine substitution, as observed in some fungal species.

Conclusions:

  • The upstream cytosine binding pocket is a significant contributor to the functional diversity of PUF RNA-binding proteins.
  • This structural feature allows for enhanced RNA specificity and plays a role in biological regulation.
  • Evolutionary analysis reveals mechanisms for the loss of this specificity-enhancing pocket.

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