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Related Experiment Videos

Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

A Gil1, P A Sharp, S F Jamison

  • 1Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.

Genes & Development
|July 1, 1991
PubMed
Summary

Polypyrimidine tract binding protein (pPTB) recognizes the polypyrimidine tract in mammalian introns, impacting pre-spliceosome complex formation. This ubiquitous splicing factor, PTB, exhibits alternative splicing, producing diverse protein forms.

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Area of Science:

  • Molecular Biology
  • RNA Splicing
  • Protein Biochemistry

Background:

  • The polypyrimidine tract in mammalian introns is crucial for pre-spliceosome formation.
  • A 62-kD protein, polypyrimidine tract binding protein (pPTB), recognizes this tract.
  • Mutations affecting pPTB binding reduce pre-spliceosome efficiency.

Purpose of the Study:

  • To purify and characterize the polypyrimidine tract binding protein (pPTB).
  • To investigate the role of pPTB in pre-spliceosome complex formation.
  • To identify and analyze different forms of the PTB protein.

Main Methods:

  • Affinity chromatography using a poly(U) matrix for protein purification.
  • Peptide sequencing to isolate complementary DNA (cDNA) sequences.

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  • Sequence analysis of isolated cDNAs to identify protein variants.

    • Main Results:

    • Purified pPTB protein to homogeneity.
    • Isolated cDNAs encoding PTB, revealing alternative splicing.
    • Identified three classes of PTB cDNAs encoding distinct protein isoforms.
    • PTB is likely a ubiquitous splicing factor expressed across cell types.
    • pPTB is distantly related to hnRNP L, suggesting a novel RNA-binding protein family.

    Conclusions:

    • Polypyrimidine tract binding protein (pPTB) is essential for pre-spliceosome formation.
    • Alternative splicing of PTB pre-mRNA generates multiple protein isoforms.
    • PTB represents a novel family of RNA-binding proteins involved in splicing.