AGO-CLIP

AGO-CLIP

Argonaute – crosslinking and immunoprecipitation (AGO-CLIP ) is a protocol that maps miRNA binding to AGO sites in C. elegans. In this method, photoreactive nucleoside 4-thiouridine (4SU) is first incorporated into C.elegans RNA in vivo. The 4SU labeled RNA is crosslinked to bound protein. The extracted lysate is treated with RNase T1, which shortens some miRNAs. After immunoprecipitation and washing, crosslinked RNA is treated with PNK, T4-Ligase and finally proteinase K. The RNA is lastly extracted, reverse transcribed to cDNA and sequenced. Deep sequencing provides single base pair resolution of miRNA and AGO binding sites.

• AGO-CLIP: Grosswendt S., Filipchyk A., Manzano M., Klironomos F., Schilling M., et al. (2014) Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell 54: 1042-1054

1. Imig J., Brunschweiger A., Brummer A., Guennewig B., Mittal N., et al. (2015) miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat Chem Biol 11: 107-114
2. Luna J. M., Scheel T. K., Danino T., Shaw K. S., Mele A., et al. (2015) Hepatitis C virus RNA functionally sequesters miR-122. Cell 160: 1099-1110
3. Wang P., Ning S., Zhang Y., Li R., Ye J., et al. (2015) Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer. Nucleic Acids Res 43: 3478-3489
4. Grosswendt S., Filipchyk A., Manzano M., Klironomos F., Schilling M., et al. (2014) Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell 54: 1042-1054
5. Newie I., Sokilde R., Persson H., Grabau D., Rego N., et al. (2014) The HER2-encoded miR-4728-3p regulates ESR1 through a non-canonical internal seed interaction. PLoS One 9: e97200