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Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

S Guth1, T Ø Tange, E Kellenberger

  • 1Gene Expression Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

Molecular and Cellular Biology
|October 18, 2001
PubMed

Insights

The splicing factor U2AF(35) plays a dual role in pre-mRNA splicing, stabilizing U2AF(65) binding and triggering spliceosome assembly. Its function is linked to AG-dependent splicing and the 3' splice site, independent of SR protein interactions.

Area of Science:

  • Molecular Biology
  • RNA Splicing Mechanisms

Background:

  • U2AF is crucial for U2 small nuclear RNP recruitment to pre-mRNAs in eukaryotes.
  • U2AF(65) binds polypyrimidine tracts, while U2AF(35) interacts with the 3' splice site AG.
  • U2AF(35) can stabilize U2AF(65) binding to weak polypyrimidine tracts and mediate interactions with SR proteins.

Purpose of the Study:

  • To investigate sequence elements governing U2AF(35) dependence in mouse immunoglobulin M (IgM) pre-mRNA splicing.
  • To elucidate the specific functions of U2AF(35) beyond stabilizing U2AF(65) binding.

Main Methods:

  • Oligo(dT)-cellulose chromatography to deplete nuclear extracts of U2AF.
  • Pre-mRNA splicing complementation assays using depleted nuclear extracts.
  • Analysis of sequence elements including polypyrimidine tract strength, 3' splice site, and exonic splicing enhancers (ESE).

Main Results:

  • IgM pre-mRNA is AG-dependent, and U2AF(35) dependence correlates with this AG dependence.
  • The first nucleotide of exon 2 is critical for U2AF(35) function.
  • RS domain-mediated interactions with SR proteins are dispensable for U2AF(35) activity in this context.

Conclusions:

  • U2AF(35) has a dual function in pre-mRNA splicing: stabilizing U2AF(65) binding and initiating spliceosome assembly events.
  • U2AF(35) interaction with the consensus 3' splice site is key to its function.
  • The findings reveal novel aspects of U2AF(35)'s role in splicing regulation.

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