Abstract
MicroRNAs (miRNAs) are small noncoding RNAs that regulate the stability and expression of target RNAs in a sequence-dependent manner. Identifying miRNA-regulated genes is key to understanding miRNA function. Here, we describe an unbiased biochemical pulldown method to identify with high-specificity miRNA targets. Regulated transcripts are enriched in streptavidin-captured mRNAs that bind to a transfected biotinylated miRNA mimic. The method is relatively simple, does not involve cross-linking and can be performed with only a million cells. Addition of an on-bead RNase digestion step also identifies miRNA recognition elements (MRE).
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References
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20
Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B, Rigoutsos I (2006) A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 126:1203–1217
Thomas M, Lieberman J, Lal A (2010) Desperately seeking microRNA targets. Nat Struct Mol Biol 17:1169–1174
Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486
Loeb GB, Khan AA, Canner D, Hiatt JB, Shendure J, Darnell RB, Leslie CS, Rudensky AY (2012) Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol Cell 48:760–770
Zisoulis DG, Lovci MT, Wilbert ML, Hutt KR, Liang TY, Pasquinelli AE, Yeo GW (2010) Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat Struct Mol Biol 17:173–179
Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141
Helwak A, Kudla G, Dudnakova T, Tollervey D (2013) Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153:654–665
Grosswendt S, Filipchyk A, Manzano M, Klironomos F, Schilling M, Herzog M, Gottwein E, Rajewsky N (2014) Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell 54:1042–1054
Keene JD, Komisarow JM, Friedersdorf MB (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc 1:302–307
Lal A, Thomas MP, Altschuler G, Navarro F, O'Day E, Li XL, Concepcion C, Han YC, Thiery J, Rajani DK et al (2011) Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet 7:e1002363
Tan SM, Kirchner R, Jin J, Hofmann O, McReynolds L, Hide W, Lieberman J (2014) Sequencing and systems analysis of captive target transcripts identifies primate-specific miR-522 as an inducer of mesenchymal transition. Cell Rep 8:1225–1239
Perdigao-Henriques R, Petrocca F, Altschuler G, Thomas MP, Le MT, Tan SM et al. miR-200 promotes the mesenchymal to epithelial transition by suppressing multiple members of the Zeb2 and Snail1 transcriptional repressor complexes. Oncogene 2015. Advanced doi: 10.1038/onc.2015.69.online publicaton
Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30:460–471
Cloonan N, Wani S, Xu Q, Gu J, Lea K, Heater S, Barbacioru C, Steptoe AL, Martin HC, Nourbakhsh E et al (2011) MicroRNAs and their isomiRs function cooperatively to target common biological pathways. Genome Biol 12:R126
Kang H, Davis-Dusenbery BN, Nguyen PH, Lal A, Lieberman J, Van Aelst L, Lagna G, Hata A (2012) Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins. J Biol Chem 287:3976–3986
Le MT, Shyh-Chang N, Khaw SL, Chin L, Teh C, Tay J, O'Day E, Korzh V, Yang H, Lal A et al (2011) Conserved regulation of p53 network dosage by microRNA-125b occurs through evolving miRNA-target gene pairs. PLoS Genet 7:e1002242
Martin HC, Wani S, Steptoe AL, Krishnan K, Nones K, Nourbakhsh E, Vlassov A, Grimmond SM, Cloonan N (2014) Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs. Genome Biol 15:R51
Tay Y, Rinn J, Pandolfi PP (2014) The multilayered complexity of ceRNA crosstalk and competition. Nature 505:344–352
Guo YE, Steitz JA (2014) 3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells. RNA 20:985–988
Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14:447–459
Acknowledgements
We thank Jingmin Jin and Larry McReynolds for their sequencing expertise, Rory Kirchner, Oliver Hofmann and Winston Hide for their bioinformatics expertise, and members of the Lieberman lab for critical discussions. S.M.T. was supported by the Department of Defense (DOD) Breast Cancer Research Program (BCRP).
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Tan, S.M., Lieberman, J. (2016). Capture and Identification of miRNA Targets by Biotin Pulldown and RNA-seq. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 1358. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3067-8_13
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DOI: https://doi.org/10.1007/978-1-4939-3067-8_13
Publisher Name: Humana Press, New York, NY
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