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. 2007 Aug;13(8):1198-204.
doi: 10.1261/rna.563707. Epub 2007 Jun 25.

Isolation of microRNA targets by miRNP immunopurification

George Easow  1 , Aurelio A Teleman, Stephen M Cohen
Affiliations

Isolation of microRNA targets by miRNP immunopurification

George Easow et al. RNA. 2007 Aug.

Abstract

microRNAs (miRNAs) serve as post-transcriptional regulators of gene expression, by guiding effector complexes (miRNPs) to target RNAs. Although considerable progress has been made in computational methods to identify miRNA targets, only a relatively limited assessment of their ability to function in vivo has been reported. Here we describe an alternative approach to miRNA target identification based on a biochemical method for purifying miRNP complexes with associated miRNAs and bound mRNA targets. Microarray analysis revealed a high degree of enrichment for miRNA complementary sites in the 3'UTRs of the miRNP-associated mRNAs. mRNAs specifically associated with an individual miRNA were identified by comparing the miRNP-associated mRNAs from wild-type flies and mutant flies lacking miR-1, and their regulation by the miRNA was validated. This approach provides a means to identify functional miRNA targets based on their physical interaction in vivo.

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Figures

FIGURE 1.
FIGURE 1.
miRNP complex purification. (A) Schematic representation of the purification protocol. F/H-dAgo1 indicates Drosophila Ago1 protein tagged at the N terminus with a FLAG epitope followed by an HA epitope. Although two epitope tags were incorporated in the Ago-1 construct, we found that the specific enrichment of Ago-containing complexes and miRNAs was not significantly improved using a two-step purification. (B) Immunoblot to visualize F/H-dAgo1 purification. Lanes labeled + contain samples from cells stably transfected to express F/H-dAgo1. The lane labeled − contains sample from untransfected S2 cells. The input and eluate samples were from the same gel, but intervening lanes are not shown. The blot was first probed with anti-HA and reprobed with anti-tubulin. (C) Northern blot probed to visualize bantam miRNA and miR-2b. Ago1 denotes eluate from F/H-dAgo1 transfected cells; cont. denotes eluate from untransfected cells. (D) Quantitative RT-PCR to detect reaper (rpr) and CG1969 mRNAs in the eluate fraction, normalized to rp49. “1” and “2” are independent purifications.
FIGURE 2.
FIGURE 2.
Enrichment of sequences complementary to miRNA seeds in Ago1 immunoprecipitates. (A, top) Northern blots probed to visualize miR-1, miR-184, miR-7, and miR-314 in total RNA from S2 cells. (Bottom) Relative enrichment of 7mer sequences complementary to positions 2–8 of the indicated miRNAs in the messenger RNAs immunopurified with Ago1. mRNA indicates the sequence matches found in the whole transcript. ORF indicates matches found in coding sequence. UTR indicates sequence matches found in 3′UTRs. Enrichment is normalized to the overall frequency of occurrence of the sequences in the mRNA, ORF, and UTR data sets. (B, top) Predicted miR-184 sites in the ORFs of CG5704 and CG1857 (top, strand mRNA; bottom, strand miR-184). Arrows indicate residues altered in the mutant constructs to disrupt pairing to miR-184, while retaining the ORF in the mRNA. The ORFs of the wild-type and mutant forms of CG5704 and CG1857 were cloned in frame with firefly luciferase. (Bottom) Luciferase assays comparing regulation by endogenous miR-184 in S2 cells transfected to express the ORF–luciferase fusion proteins. Levels were normalized to the wild-type construct. Elevated activity in the mutant constructs indicates alleviation of repression mediated by miR-184. (C) Luciferase assays for regulation of the reporter constructs depicted at left by miR-278. SV40 and expanded indicate the origin of the 3′UTR. Colored arrowheads indicate the introduced miR-278 sites. Boxes at right show these sites: “ex” shows the endogenous miR-278 site in the expanded 3′UTR; black triangle shows a synthetic miR-278; green and red triangles denote the alteration of the luciferase ORF to produce sites matching the miR-278 seed region.
FIGURE 3.
FIGURE 3.
Alterations in the profile of Ago1-associated RNAs by adding or removing miR-1. (A, left) Northern blot comparing miR-1 expression in untransfected (−) and transfected (+) S2 cells. (Right) Enrichment of 7mer sequences complementary to positions 2–8 of miR-1 or miR-92b in RNA copurified with Ago1. (B) Enrichment of 7mer sequences complementary to miR-1 in total RNA copurified with Ago1 from control or miR-1 mutant embryos. miR-1(1) indicates complementarity to positions 1–7; mir-1(2) to positions 2–8. GU(#) indicates sequence matches with a GU base pair at the indicated position.
FIGURE 4.
FIGURE 4.
mRNAs differentially enriched in control and miR-1 mutant embryos. (A) Histogram showing regulation by miR-1 of luciferase reporters containing the 3′UTRs of the 11 mRNAs that contributed to the enrichment signal from panel B. All were down-regulated in cells transfected to express miR-1 compared with control cells. A Renilla luciferase construct with the sv40 3′UTR was cotransfected to provide a normalization control. (B) Immunoblot of proteins samples from 18- to 24-h control embryos (wt) and miR-1 mutant embryos (miR-1KO), probed with antibody to Nedd4 and then reprobed with anti-kinesin as a loading control.
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