The key feature of the methodology is the induction of covalent cross-links between an RNA-binding protein and its directly bound RNA molecules (within ~1 Å) by UV irradiation 8. Therefore, an important step towards understanding RNA regulation at the molecular level is to reveal the positional information about the binding sites of RBPs.Ī strategy referred to as cross-linking and immunoprecipitation (CLIP-seq) has been developed to capture protein-RNA interactions with UV cross-linking followed by immunoprecipitation of the protein of interest 7.
The delicate mechanisms of these processes depend on the unique spatial and temporal arrangement of RNAs and RBPs. RNAs and RNA-binding proteins (RBPs) control diverse cellular processes including splicing, translation, ribosome biogenesis, epigenetic regulation, and cell fate transition 1, 2, 3, 4, 5, 6. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification.
Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. RNA and RNA-binding proteins (RBPs) control multiple biological processes.
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