In vivo identification of ribonucleoprotein-RNA interactions.
To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.
Zielinski
Jennifer
J
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Kilk
Kalle
K
Peritz
Tiina
T
Kannanayakal
Theresa
T
Miyashiro
Kevin Y
KY
Eiríksdóttir
Emelía
E
Jochems
Jeanine
J
Langel
Ulo
U
Eberwine
James
J
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D017403:In Situ Hybridization; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D051381:Rats; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays
D000596:Amino Acids; D003432:Cross-Linking Reagents; D017931:DNA Primers; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D010455:Peptides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA; D004247:DNA