RNA-Protein Interaction Protocols
Due to the vital biological importance of RNA and proteins functioning together within a cell, a protocol volume describing experimental procedures to study their interactions should find a home in many laboratories. RNA-Protein Interaction Protocols, Sec
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n Those interested in the role RNA binding proteins play in the development and function of organisms have been limited by a paucity of techniques available to study RNA–protein interactions in vivo. Conversely, biochemists have developed potent methods for studying interactions in vitro but have not applied these to living tissues. We describe here a method in which a classical in vitro tool used by RNA biochemists—ultraviolet (UV) crosslinking—is adapted to the study of RNA–protein complexes in living tissues. We have successfully From: Methods in Molecular Biology, vol. 488: RNA-Protein Interaction Protocols Edited by: Ren-Jang Lin © Humana Press Inc, Totowa, NJ
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Jensen and Darnell
used this method, termed CLIP (crosslinking and immunoprecipitation of RNA–protein complexes) to identify a number of target RNAs of the Nova family of neuron-specific RNA binding proteins (1,2), and we are piloting its use with several other RNA binding proteins of interest in our laboratories, notably the FMRP and Hu families. The term CLIP is largely self-explanatory, with the following addendums: We have used a living organ—specifically brain tissue—as the source for in vivo RNA–protein complexes to be CLIPed. This tissue is dissected from the animal (mouse in our experiments), rapidly dissociated on ice by trituration to allow UV light to penetrate the cells, and irradiated immediately thereafter. A series of subsequent biochemical steps is used to partially hydrolyze the RNA, allowing short (~70- to 100-nt) fragments to remain bound to the protein. We have used ribonuclease (RNase) T1, which leaves a 5' –OH and a 2',3'-cyclic phosphate on the RNA chain. The complex is then purified by immunoprecipitation, a step obviously critical to the protocol and dependent on an antibody good for immunoprecipitation. The RNA–protein complex is then treated with T4 polynucleotide kinase (PNK; which both adds a 5'-PO4− group and resolves the 2',3'-cyclic phosphate to a 3' –OH), a step that allows the introduction of radioactive tracer and prepares the RNA ends for subsequent directional linker ligation. The complex is then further purified by boiling in sodium dodecyl sulfate (SDS)-sample buffer and running on denaturing SDS polyacrylamide gel electrophoresis (PAGE) gels. This gel purification step is critical to the success of CLIP because it partitions the RNA–protein covalent complex away from any RNA that has immunoprecipitated with the protein but was not crosslinked to the protein. While this “free” RNA pool may contain bone fide RNA targets of the protein, it may also include RNAs that have only bound the protein in vitro during the purification steps of the protocol, and thus it is critical to remove these RNAs (3). A final purification step is achieved by transferring the gel to nitrocellulose; this transfer allows purification of RNA–protein complexes from the membrane (much easier than from the gel matrix itself), and at the same time, any remaining free RNA in the gel passes through the nitrocellulose and toward the positiv
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