Article: Ribonucleoprotein immunoprecipitation (RNP-IP): a direct in vivo analysis of microRNA-targets

We have developed a novel Ribonucleoprotein Immunoprecipitation (RNP-IP) method to isolate miR-RISC complexes, associated microRNAs and target mRNAs. Our method characterizes mRNAs present in immunoprecipitates containing miR-RISC complexes that were obtained using GW182 and AGO2 antibodies. MicroRNA bound transcripts were reverse transcribed and amplified using seed sequence and 3'UTR derived primers. This flexible IP-based assay is a straightforward method to identify miRs participating in gene regulation and their cognate mRNAs in real time.

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References

1.

Pillai R, Bhattacharyya S, Filipowicz W . Repression of protein synthesis by miRNAs: how many mechanisms?. Trends Cell Biol. 2007; 17(3):118-26. DOI: 10.1016/j.tcb.2006.12.007. View

2.

Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T . Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15(2):185-97. DOI: 10.1016/j.molcel.2004.07.007. View

3.

Pillai R . MicroRNA function: multiple mechanisms for a tiny RNA?. RNA. 2005; 11(12):1753-61. PMC: 1370863. DOI: 10.1261/rna.2248605. View

4.

Yang Z, Edenberg H, Davis R . Isolation of mRNA from specific tissues of Drosophila by mRNA tagging. Nucleic Acids Res. 2005; 33(17):e148. PMC: 1243647. DOI: 10.1093/nar/gni149. View

5.

Keene J, Komisarow J, Friedersdorf M . RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc. 2007; 1(1):302-7. DOI: 10.1038/nprot.2006.47. View

6.

Gerber A, Luschnig S, Krasnow M, Brown P, Herschlag D . Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2006; 103(12):4487-92. PMC: 1400586. DOI: 10.1073/pnas.0509260103. View

7.

Brown V, Jin P, Ceman S, Darnell J, ODonnell W, Tenenbaum S . Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell. 2001; 107(4):477-87. DOI: 10.1016/s0092-8674(01)00568-2. View

8.

Tenenbaum S, Carson C, Lager P, Keene J . Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A. 2000; 97(26):14085-90. PMC: 18875. DOI: 10.1073/pnas.97.26.14085. View

9.

Liu J, Carmell M, Rivas F, Marsden C, Thomson J, Song J . Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004; 305(5689):1437-41. DOI: 10.1126/science.1102513. View

10.

Gerber A, Herschlag D, Brown P . Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2004; 2(3):E79. PMC: 368173. DOI: 10.1371/journal.pbio.0020079. View

11.

Zhang L, Ding L, Cheung T, Dong M, Chen J, Sewell A . Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol Cell. 2007; 28(4):598-613. PMC: 2186060. DOI: 10.1016/j.molcel.2007.09.014. View

Ribonucleoprotein Immunoprecipitation (RNP-IP): a Direct in Vivo Analysis of MicroRNA-targets