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Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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Distinct roles of Pcf11 zinc-binding domains in pre-mRNA 3'-end processing

Julia Guéguéniat1, Adrien F Dupin1, Johan Stojko2

  • 1Université de Bordeaux, INSERM U1212, CNRS UMR5320, Bordeaux, France.

Nucleic Acids Research
|October 4, 2017
PubMed

Insights

Researchers identified two zinc-binding domains in the Pcf11 protein, a key component of yeast

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Messenger RNA (mRNA) biogenesis involves crucial 3'-end processing steps, including pre-mRNA cleavage and polyadenylation.
  • The yeast Cleavage Factor IA (CF IA) complex, comprising Pcf11, Clp1, Rna14, and Rna15, orchestrates these 3'-end processing events.
  • The Pcf11 protein's structure and specific functional domains within CF IA remain incompletely understood.

Purpose of the Study:

  • To elucidate the structural characteristics of the Pcf11 protein, focusing on its zinc-binding domains.
  • To investigate the role of these zinc-binding domains in CF IA assembly, transcription termination, and pre-mRNA 3'-end processing.
  • To enhance the understanding of Pcf11's architecture and its functional contribution to mRNA maturation in yeast.

Main Methods:

  • Structural characterization of Pcf11, including the identification and analysis of its zinc-binding domains.
  • Biochemical assays to assess the binding of Zn2+ ions to Pcf11.
  • Functional studies to evaluate the necessity of the zinc-binding domains for CF IA assembly, transcription termination, and pre-mRNA processing.

Main Results:

  • Evidence for the binding of two Zn2+ atoms to Pcf11, each coordinated by distinct zinc-binding domains.
  • Structural determination of one zinc-binding domain revealing an unusual zinc finger fold.
  • Demonstration that these zinc-binding domains are not essential for CF IA assembly or transcription termination but play roles in 3'-end processing.

Conclusions:

  • The Pcf11 protein possesses two distinct zinc-binding domains, one of which exhibits an atypical zinc finger structure.
  • These domains are dispensable for the assembly of the CF IA complex and RNA polymerase II transcription termination.
  • The zinc-binding domains contribute to the pre-mRNA 3'-end processing mechanism, highlighting their specific functional importance.

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