PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): A Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins

Methods in Enzymology

Volumn 539, Issue , 2014, Pages 113-161

  Spitzer, Jessica ^a   Hafner, Markus ^a   Landthaler, Markus ^c   Ascano, Manuel ^a   Farazi, Thalia ^a   Wardle, Greg ^a   Nusbaum, Jeff ^a   Khorshid, Mohsen ^b   Burger, Lukas ^b   Zavolan, Mihaela ^b   Tuschl, Thomas ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

^b SWISS INSTITUTE OF BIOINFORMATICS   (Switzerland)

^c MAX DELBRÜCK CENTER FOR MOLECULAR MEDICINE   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

4 THIOURIDINE; ANTIBODY; BUFFER; DYES, REAGENTS, INDICATORS, MARKERS AND BUFFERS; MESSENGER RNA; OLIGONUCLEOTIDE; PROTEINASE K; RIBONUCLEASE T1; RNA BINDING PROTEIN; UNCLASSIFIED DRUG;

ADHERENT CELL; ANALYTICAL EQUIPMENT; ARTICLE; CELL LABELING; CELL LYSATE; CULTURE MEDIUM; DNA LIBRARY; GENE AMPLIFICATION; IMMUNOPRECIPITATION; ISOTOPE LABELING; PHOTOACTIVATABLE RIBONUCLEOSIDE ENHANCED CROSSLINKING AND IMMUNOPRECIPITATION; POLYACRYLAMIDE GEL ELECTROPHORESIS; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; PROTEIN CROSS LINKING; PROTEIN DEPHOSPHORYLATION; RNA DEGRADATION; SUSPENSION CELL CULTURE; THEORY; TRANSCRIPTOMICS;

3′- AND 5′-ADAPTER LIGATION; CDNA LIBRARY PREPARATION; CDNA LIBRARY PREPARATION/REVERSE TRANSCRIPTION; CELL LYSATE PREPARATION; CROSSLINKING AND IMMUNOPRECIPITATION (CLIP) PROTOCOL; IMMUNOPRECIPITATION AND SECOND RNASE T1 DIGESTION; PCR AMPLIFICATION OF CDNA LIBRARY; POSTTRANSCRIPTIONAL REGULATION (PTR); PROTEIN G MAGNETIC BEADS; PROTEINASE K DIGESTION; UV CROSSLINKING OF 4-THIOURIDINE-LABELED CELLS;

ANIMALS; BINDING SITES; CELLS, CULTURED; ENDOPEPTIDASE K; HUMANS; IMMUNOPRECIPITATION; PHOTOCHEMICAL PROCESSES; PROTEOLYSIS; RNA; RNA-BINDING PROTEINS; TRANSCRIPTOME; ULTRAVIOLET RAYS;

EID: 84899667216     PISSN: 00766879     EISSN: 15577988     Source Type: Book Series    
DOI: 10.1016/B978-0-12-420120-0.00008-6     Document Type: Chapter

References (16)

1. Ambros, V. (2004). The functions of animal microRNAs. Nature, 431(7006), 350-355.
2. Andrus, A., & Kuimelis, R. G. (2001). Base composition analysis of nucleosides using HPLC. In A. Andrus & R. G. Kuimelis (Eds.), Current Protoccols Nucleic Acid Chemistry. New York: Wiley Unit 10 16.
3. Bezerra, R., & Favre, A. (1990). In vivo incorporation of the intrinsic photolabel 4-thiouridine into Escherichia coli RNAs. Biochemical and Biophysical Research Communications, 166(1), 29-37.
4. Favre, A., Moreno, G., Blondel, M. O., Kliber, J., Vinzens, F., & Salet, C. (1986). 4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells. Biochemical and Biophysical Research Communications, 141(2), 847-854.
5. Gerber, A. P., Luschnig, S., Krasnow, M. A., Brown, P. O., & Herschlag, D. (2006). Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America, 103(12), 4487-4492.
6. Glisovic, T., Bachorik, J. L., Yong, J., & Dreyfuss, G. (2008). RNA-binding proteins and post-transcriptional gene regulation. FEBS Letters, 582(14), 1977-1986.
7. Halbeisen, R. E., Galgano, A., Scherrer, T., & Gerber, A. P. (2008). Post-transcriptional gene regulation: From genome-wide studies to principles. Cellular and Molecular Life Sciences, 65(5), 798-813.
8. Hafner, M., Landgraf, P., Ludwig, J., et al. (2008). Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods, 44(1), 3-12.
9. Hafner, M., Landthaler, M., Burger, L., et al. (2010). PAR-CLIP-Transcriptome-wide identification of RNA targets and binding sites of RNA-binding proteins. Cell, 141(1), 129-141.
10. Keene, J. D. (2007). RNA regulons: Coordination of post-transcriptional events. Nature Reviews Genetics, 8(7), 533-543.
11. Landthaler, M., Gaidatzis, D., Rothballer, A., et al. (2008). Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA, 14(12), 2580-2596.
12. Moore, M. J. (2005). From birth to death: The complex lives of eukaryotic mRNAs. Science, 309(5740), 1514-1518.
13. Tenenbaum, S. A., Carson, C. C., Lager, P. J., & Keene, J. D. (2000). Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proceedings of the National Academy of Sciences of the United States of America, 97(26), 14085-14090.
14. Ule, J., Jensen, K. B., Ruggiu, M., Mele, A., Ule, A., & Darnell, R. B. (2003). CLIP identifies Nova-regulated RNA networks in the brain. Science, 302(5648), 1212-1215.
15. Hafner, M., Landgraf, P., Ludwig, J., et al. (2008). Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods, 44(1), 3-12.
16. Hafner, M., Landthaler, M., Burger, L., et al. (2010). PAR-CLIP-Transcriptome-wide identification of RNA targets and binding sites of RNA-binding proteins. Cell, 141(1), 129-141.