Karginov, F. V., Conaco, C., Xuan, Z. Y., Schmidt, B. H., Parker, J. S., Mandel, G., Hannon, G. J. (December 2007) A biochemical approach to identifying microRNA targets. Proceedings of the National Academy of Sciences of the United States of America, 104 (49). pp. 19291-6. ISSN 0027-8424
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Abstract
Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.
| Item Type: | Paper |
|---|---|
| Additional Information: | |
| Uncontrolled Keywords: | gene regulation RNAi RISC immunoprecipitation |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA |
| CSHL Authors: | |
| Communities: | CSHL labs > Hannon lab CSHL Post Doctoral Fellows |
| Depositing User: | CSHL Librarian |
| Date: | 4 December 2007 |
| Date Deposited: | 11 Nov 2011 16:34 |
| Last Modified: | 05 Jan 2018 20:26 |
| PMCID: | PMC2148283 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/23058 |
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