Title | High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Li X, Pritykin Y, Concepcion CP, Lu Y, La Rocca G, Zhang M, King B, Cook PJ, Au YWah, Popow O, Paulo JA, Otis HG, Mastroleo C, Ogrodowski P, Schreiner R, Haigis KM, Betel D, Leslie CS, Ventura A |
Journal | Mol Cell |
Volume | 79 |
Issue | 1 |
Pagination | 167-179.e11 |
Date Published | 2020 Jul 02 |
ISSN | 1097-4164 |
Keywords | Animals, Argonaute Proteins, Embryonic Stem Cells, Female, Gene Expression Regulation, Glioma, High-Throughput Nucleotide Sequencing, Hydrolases, Mice, Mice, Knockout, MicroRNAs, Protein Binding, Recombinant Fusion Proteins, RNA, Messenger |
Abstract | <p>The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.</p> |
DOI | 10.1016/j.molcel.2020.05.009 |
Alternate Journal | Mol Cell |
PubMed ID | 32497496 |
PubMed Central ID | PMC7446397 |
Grant List | 1F31CA168356-01A1 / CA / NCI NIH HHS / United States F31 CA168356 / CA / NCI NIH HHS / United States P30 CA008748 / CA / NCI NIH HHS / United States R01 CA149707 / CA / NCI NIH HHS / United States R01 CA245507 / CA / NCI NIH HHS / United States |