Reprogramming Alternative Pre-Messenger RNA Splicing Through the Use of Protein-Binding Antisense Oligonucleotides

J Biol Chem. 2003 Dec 12;278(50):50031-9. doi: 10.1074/jbc.M308897200. Epub 2003 Sep 30.

Abstract

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Dose-Response Relationship, Drug
  • Exons
  • Glutathione Transferase / metabolism
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Oligonucleotides / chemistry
  • Oligonucleotides, Antisense / chemistry*
  • Plasmids / metabolism
  • Protein Binding
  • RNA Splicing*
  • RNA, Messenger / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ribonuclease H / metabolism
  • Ribonucleoprotein, U1 Small Nuclear / metabolism
  • Transcription, Genetic
  • Transfection

Substances

  • Oligonucleotides
  • Oligonucleotides, Antisense
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Ribonucleoprotein, U1 Small Nuclear
  • Glutathione Transferase
  • Ribonuclease H