Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides.
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.
Villemaire
Jonathan
J
Département de microbiologie et d'infectiologie, RNA/RNP Group, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Dion
Isabelle
I
Elela
Sherif Abou
SA
Chabot
Benoit
B
D017398:Alternative Splicing; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005091:Exons; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D005982:Glutathione Transferase; D016914:Ribonuclease H