CLIP: a method for identifying protein-RNA interaction sites in living cells.
Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.
Ule
Jernej
J
Howard Hughes Medical Institute and the Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, USA.
Jensen
Kirk
K
Mele
Aldo
A
Darnell
Robert B
RB
D000818:Animals; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D005784:Gene Amplification; D047468:Immunoprecipitation; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins