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Analysis of protein/RNA-interactions using iCLIP

...we are using crosslinking & immunoprecipitation analyses to track transcriptome-wide protein/RNA interactions


König, ...Ule (2010) Nat Struct Mol Biol, modified
Buchbender, ...König (2020) Methods

 

Every 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. 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.

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.

In iCLIP, (1) live cultured cells are irradiated with UV light to crosslink RNA binding proteins to cellular RNA. (2) After cell lysis, the RNA is trimmed by limited RNase digestion. (3) The protein of interest (X) is immunoprecipitated with a specific antibody. (4) The cyclic phosphate produced by RNase digestion is removed by phosphatase treatment. (5) An RNA linker is ligated to the 3’ end of the RNA. (6) The RNA is radioactively 5’ end-labeled with ³²P. (7) Free RNA is removed by gel electrophoresis followed by transfer to a nitrocellulose membrane, which binds proteins unspecifically. After autoradiography, the area with the covalent protein/RNA complexes of interest is cut from the membrane. (8) The RNA is eluted from the membrane by protein digestion with proteinase K. Note that a single amino acid remains at the crosslink site. (9) The RNA is reverse-transcribed and in most cases, the reverse transcriptase stops one nucleotide prior to the crosslink site. (10) The cDNA is ligated at it now 3'-end with a DNA oligonucleotide that introduces additional sequences. The region depicted in blue is compatible with high-throughput sequencing, and in addition a random and an experimental barcode are added. (11) The cDNA is amplified by PCR with primers compatible with high-throughput sequencing. Note that the 5’ end of the PCR product (excluding linker sequence) marks the initial crosslink site. (12) The PCR product pool is subjected to high-throughput sequencing. From Wang et al (2009) and König et al (2010), modified.

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. iCLIP data sets can be used to screen for novel RNA interactors of a specific RBP and unravel yet unknown functions in regulatory networks. 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.

 

  • Klein T, Funke F, Rossbach O, Lehmann G, Vockenhuber M, Medenbach J, Suess B, Meister G, Babinger P. Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli. International Journal of Molecular Sciences. 2023 Jun 16; 24(14):11536. (Article Link)
  • Stebel S, Breuer J, Rossbach O. Studying miRNA–mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein. Methods and Protocols. 2022 Nov 30;5(6):96. (Article Link)
  • 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. RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling. Cell Reports. 2022 May 31;39(9):110895. PMID: 35649367 (Article Link)
  • 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. Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26. Life Science Alliance. 2022 May 5;5(8):e202201418. PMID: 35512835 (Article Link)
  • Bathke J, Gauernack S, Rupp O, Weber L, Preusser C, Lechner M, Rossbach O, Goesmann A, Evguenieva-Hackenberg E, Klug G. iCLIP analysis of RNA substrates of the archaeal exosome. BMC Genomics. 2020 Nov 16;21(1):797. (Article Link)
  • 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. Long Noncoding RNA TYKRIL Plays a Role in Pulmonary Hypertension via the p53-Mediated Regulation of PDGFRβ. American Journal of Respiratory and Critical Care Medicine. 2020 Nov 15;202(10):1445-1457. (Article Link)
  • Kilchert C, Kecman T, Priest E, Hester S, Aydin E, Kus K, Rossbach O, Castello A, Mohammed S, Vasiljeva L. System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA-protein complexes. Genome Research. 2020 Jul;30(7):1012-1026. (Article Link)
  • Deutschmeyer V, Breuer J, Walesch SK, Sokol AM, Graumann J, Bartkuhn M, Boettger T, Rossbach O, Richter AM. Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function. Clinical Epigenetics. 2019 Dec 4;11(1):182. (Article Link)
  • Moschall R, Rass M, Rossbach O, Lehmann G, Kullmann L, Eichner N, Strauss D, Meister G, Schneuwly S, Krahn M, Medenbach J. Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by  controlling Sex-lethal alternative splicing. Nucleic Acids Research. 2019 Mar 18;47(5):2276-2288. (Article Link)
  • Neumann P, Jaé N, Knau A, Fouani Y, Rossbach O, Krüger M, John D, Bindereif A, Grote P, Boon RA, Dimmeler S. The lncRNA GATA6-AS epigenetically regulates endothelial gene expression via interaction with LOXL2. Nature Communications. 2018 Jan 16; 9(1):237. (Article Link)
  • Stellos K, Gatsiou A, Stamatelopoulos K, Perisic Matic L, John D, Lunella FF, Jaé N, Rossbach O, Amrhein C, Sigala F, Boon RA, Fürtig B, Manavski Y, You X, Uchida S, Keller T, Boeckel JN, Franco-Cereceda A, Maegdefessel L, Chen W, Schwalbe H, Bindereif A, Eriksson P, Hedin U, Zeiher AM, Dimmeler S. Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated posttranscriptional regulation. Nature Medicine. 2016 Oct; 22(10):1140-1150. (Article Link)
  • Schneider T, Hung LH, Schreiner S, Starke S, Eckhof H, Rossbach O, Reich S, Medenbach J, Bindereif A. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs. Scientific Reports. 2016 Aug 11; 6:31313. (Article Link)
  • Preußer C*, Rossbach O*, Hung LH, Li D, Bindereif A. Genome-wide RNA-binding analysis of the trypanosome U1 snRNP proteins U1C and U1-70K reveals cis/trans-spliceosomal network. Nucleic Acids Research. 2014 Jun; 42(10):6603-15. (Article Link)
  • Rossbach O, Hung LH, Khrameeva E, Schreiner S, König J, Curk T, Zupan B, Ule J, Gelfand MS, Bindereif A. Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L. RNA Biology. 2014 Feb; 11(2):146-55. (Article Link)
  • Rossbach O, Bindereif A. Unraveling RNA-mediated networks: new insights from new technologies. Biological Chemistry. 2014 Jan; 395(1):51-60. (Article Link)

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     (* these authors contributed equally)