Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing

Mol Cell. 2000 Jun;5(6):949-57. doi: 10.1016/s1097-2765(00)80260-9.

Abstract

We studied the role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. We conclude that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Binding Sites
  • Cell Extracts
  • Exons
  • Genes, src / genetics*
  • HeLa Cells
  • Humans
  • Introns
  • Mutation
  • Neurons / cytology
  • Neurons / metabolism
  • Organ Specificity
  • Polypyrimidine Tract-Binding Protein
  • Protein Binding / drug effects
  • Protein Binding / radiation effects
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / immunology
  • RNA-Binding Proteins / metabolism*
  • Regulatory Sequences, Nucleic Acid / genetics
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Ribonucleoproteins / immunology
  • Ribonucleoproteins / metabolism*
  • Spliceosomes / metabolism
  • Tumor Cells, Cultured
  • Ultraviolet Rays

Substances

  • Cell Extracts
  • RNA Precursors
  • RNA, Messenger
  • RNA-Binding Proteins
  • Ribonucleoprotein, U2 Small Nuclear
  • Ribonucleoproteins
  • Polypyrimidine Tract-Binding Protein
  • Adenosine Triphosphate