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Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65

E Allen Sickmier1, Katherine E Frato, Haihong Shen

  • 1Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA.

Molecular Cell
|July 5, 2006
PubMed

Insights

The U2AF(65) protein recognizes specific RNA sequences crucial for pre-mRNA splicing. Its structure reveals how it binds uridine-rich tracts while allowing for variations in natural sequences.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • RNA Processing

Background:

  • U2AF(65) is a key factor in pre-mRNA splicing, recognizing polypyrimidine (Py) tracts near the 3' splice site.
  • The variable conservation of Py tracts in eukaryotes presents a challenge for U2AF(65) in recognizing uridine-rich sequences while tolerating nucleotide substitutions.

Purpose of the Study:

  • To elucidate the structural basis of U2AF(65)'s RNA binding characteristics.
  • To understand how U2AF(65) recognizes polyuridine tracts and accommodates sequence variations.

Main Methods:

  • X-ray crystallography of the U2AF(65) RNA binding domain complexed with a polyuridine tract.
  • Site-directed mutagenesis of U2AF(65) and surface plasmon resonance (SPR) analysis to assess binding energetics.

Main Results:

  • The crystal structure at 2.5 Å resolution reveals specific hydrogen bonds between U2AF(65) and uracil bases, explaining polyuridine recognition.
  • Flexible side chains and water molecules mediate most base contacts, suggesting adaptability to different Py tract sequences.
  • SPR analysis confirmed the energetic importance of conserved residues for Py tract binding.

Conclusions:

  • The study provides a structural explanation for U2AF(65)'s recognition of polyuridine tracts.
  • The findings suggest a mechanism for U2AF(65) to bind conserved uridine-rich sequences while tolerating variations in natural polypyrimidine tracts.
  • Mutational analysis validates the role of specific residues in U2AF(65)-RNA interactions.

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