Deutschmeyer V, Breuer J, Walesch SK, Sokol AM, Graumann J, Bartkuhn M, Boettger T, Rossbach O, Richter AM.System-wide analyses of the fission yeast poly(A) + RNA interactome reveal insights into organization and function of RNA-protein complexes. Genome Research. Kilchert C, Kecman T, Priest E, Hester S, Aydin E, Kus K, Rossbach O, Castello A, Mohammed S, Vasiljeva L.American Journal of Respiratory and Critical Care Medicine. Long Noncoding RNA TYKRIL Plays a Role in Pulmonary Hypertension via the p53-Mediated Regulation of PDGFRβ. Zehendner CM, Valasarajan C, Werner A, Boeckel JN, Bischoff FC, John D, Weirick T, Glaser SF, Rossbach O, Jaé N, Demolli S, Khassafi F, Yuan K, de Jesus Perez VA, Michalik KM, Chen W, Seeger W, Guenther A, Wasnick RM, Uchida S, Zeiher AM, Dimmeler S, Pullamsetti SS.iCLIP analysis of RNA substrates of the archaeal exosome. Bathke J, Gauernack S, Rupp O, Weber L, Preusser C, Lechner M, Rossbach O, Goesmann A, Evguenieva-Hackenberg E, Klug G.Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26. Salerno-Kochan A, Horn A, Ghosh P, Nithin C, Kościelniak A, Meindl A, Strauss D, Krutyhołowa R, Rossbach O, Bujnicki JM, Gaik M, Medenbach J, Glatt S.RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling. Ullah I, Thölken C, Zhong Y, John M, Rossbach O, Lenz J, Gößringer M, Nist A, Albert L, Stiewe T, Hartmann R, Vázquez O, Chung HR, Mackay JP, Brehm A.Studying miRNA–mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein. In combination with stimuli that change the cell physiology, such as hypoxia, immune stimulation, viral or bacterial infection or simply the knockdown of another factor of interest, iCLIP is powerful tool to analyze the changes in protein/RNA interactions relevant in the model system and shed light on yet unknown networks and regulatory implications of RNA-binding proteins. iCLIP data sets can be used to screen for novel RNA interactors of a specific RBP and unravel yet unknown functions in regulatory networks. The obtained sequences represent the pool of RNA molecules bound to the RBP in the living cell at the moment of the initial crosslink, with the 5’-end representing the crosslink site. From Wang et al (2009) and König et al (2010), modified. (12) The PCR product pool is subjected to high-throughput sequencing. Note that the 5’ end of the PCR product (excluding linker sequence) marks the initial crosslink site. (11) The cDNA is amplified by PCR with primers compatible with high-throughput sequencing. The region depicted in blue is compatible with high-throughput sequencing, and in addition a random and an experimental barcode are added. (10) The cDNA is ligated at it now 3'-end with a DNA oligonucleotide that introduces additional sequences. (9) The RNA is reverse-transcribed and in most cases, the reverse transcriptase stops one nucleotide prior to the crosslink site. Note that a single amino acid remains at the crosslink site. (8) The RNA is eluted from the membrane by protein digestion with proteinase K. After autoradiography, the area with the covalent protein/RNA complexes of interest is cut from the membrane.
#ICLIP EXPERIMENTS PROTOCOLS RNA IP FREE#
(7) Free RNA is removed by gel electrophoresis followed by transfer to a nitrocellulose membrane, which binds proteins unspecifically. (6) The RNA is radioactively 5’ end-labeled with ³²P. (5) An RNA linker is ligated to the 3’ end of the RNA. (4) The cyclic phosphate produced by RNase digestion is removed by phosphatase treatment.
(3) The protein of interest (X) is immunoprecipitated with a specific antibody. (2) After cell lysis, the RNA is trimmed by limited RNase digestion. In iCLIP, (1) live cultured cells are irradiated with UV light to crosslink RNA binding proteins to cellular RNA. We have established the individual-nucleotide resolution crosslinking an immunoprecipitation (iCLIP) method and applied it to a large variety of biological systems, including many different human tumor cell lines, Trypanosomes, Drosophila and Archea analyzing a variety different proteins, including several RBPs, but also RNA-interacting metabolic enzymes and chromatin-associated DNA-binding proteins that were found to also show affinity for RNA in vitro. Knowing the binding targets and the precise binding positions of the RBPs on their target transcripts is crucial for the understanding of any regulatory step of RNA processing. Ule (2010) Nat Struct Mol Biol, modifiedĮvery RNA molecule that is transcribed in the cell is immediately coated with many different RNA binding proteins (RBPs) that play a crucial role in any step of RNA processing, localization and translation.
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